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9145
Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb
Primary Antibodies
Monoclonal Antibody

Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145

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Citations (1564)
Filter:
  1. WB
  2. IP
  3. IHC
  4. IF
  5. F
  6. ChIP
Western Blotting Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Western blot analysis of extracts from IFN-alpha treated Jurkat cells and HeLa cells (left), as well as EGF treated A431 cells (right), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb. Note that the basal phospho-Stat3 in A431 is detected by the antibody.

No image available
Immunohistochemistry Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Immunohistochemical analysis of paraffin-embedded human neuroendocrine lung carcinoma using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb performed on the Leica® BOND™ Rx.

Immunohistochemistry Image 2: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb performed on the Leica® BOND™ Rx.

Immunohistochemistry Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Immunohistochemical analysis of paraffin-embedded Apc (min/+) mouse intestine, using Phosho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

Immunohistochemistry Image 2: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Immunohistochemical analysis of paraffin embedded human breast carcinoma, specifically endothelial cells, untreated (left) or lambda phosphatase treated (right), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

Immunohistochemistry Image 3: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Immunnohistochemical analysis of paraffin-embedded human lung carcinoma, showing nuclear localization, using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

Immunohistochemistry Image 4: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Immunohistochemical analysis using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb on SignalSlide® HeLa -/+ IFNa IHC Controls #55861 (paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Interferon-α1 (hIFN-α1) #8927 (right)).

Immunofluorescence Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Confocal immunofluorescent analysis of HeLa cells, IFN-alpha treated (left) or untreated (right), labeled with Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (green).

Flow Cytometry Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Flow cytometric analysis of U266 cells, untreated (blue) or treated with IFNalpha (50 ng/ml, 15 min; green) using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (solid lines) or concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Chromatin Immunoprecipitation Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes and Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IRF1, a known target gene of Phospho-Sata3 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

Chromatin Immunoprecipitation Image 2: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes, and either Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb or of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human IRF-1 promoter primers, SimpleChIP® Human c-Fos Promoter Primers #4663, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

To Purchase # 9145S
Product # Size Price
9145T
20 µl $ 138
9145S
100 µl $ 346
9145L
300 µl $ 816

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa) 79, 86
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

Application Dilution
Western Blotting 1:2000
Immunoprecipitation 1:100
IHC-Leica® Bond™ 1:100 - 1:400
Immunohistochemistry (Paraffin) 1:100 - 1:400
Immunofluorescence (Immunocytochemistry) 1:100 - 1:200
Flow Cytometry 1:100 - 1:400
Chromatin IP 1:100
Chromatin IP-seq 1:100

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 10

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.

    NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

  3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
  4. Protein A Agarose Beads: (#9863).
  5. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
  6. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

  1. Vortex to mix beads.
  2. Add 10–30 µl of 50% Protein A agarose bead slurry to 200 µl cell lysate at 1 mg/ml.
  3. Incubate with rotation at 4°C for 30–60 min.
  4. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
  5. Proceed to immunoprecipitation below.

Immunoprecipitation

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
  2. Add protein A agarose (10–30 µl of 50% bead slurry). Incubate with rotation for 1–3 hr at 4°C.
  3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
  4. Proceed to sample analysis by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15–30 µl) on SDS-PAGE (4–20%).

posted December 2008

revised April 2018

Protocol Id: 409

Immunohistochemistry (Leica® BOND™)

NOTE: Please see product datasheet or product webpage for appropriate antibody dilution^.

  Step Reagents Time/Temperature
1 Dewax BOND™ Dewax Solution, 100% Alcohol, BOND™ Wash Solution Pre-programmed Leica® BOND™ 
2 Antigen Retrieval  BOND™ Epitope Retrieval ER2 Solution 20 min., 100˚C | Protocol: HIER 20 min with ER2
3 Peroxide Block Refine Detection Kit Peroxide Block* 5 min.
  WASH BOND™ Wash Solution  3x 0:00 min.
4 Protein Block (optional) #5425 NGS or #15019 Animal-Free Blocking Solution   20 min. 
5 Primary Antibody^ Dilute in #8112 SignalStain® Antibody Diluent 30 min. 
  WASH BOND™ Wash Solution  3x 2:00 min.
NA Post Primary Mouse Linker Refine Detection Kit Post Primary*  Not Applied 
6 Secondary Detection Refine Detection Kit Polymer*  10 min. 
  WASH BOND™ Wash Solution/Deionized Water Custom (see below)
7a Visualization Refine Detection Kit Mixed DAB Refine*  0:00 min. 
7b Visualization Refine Detection Kit Mixed DAB Refine*  10 min. 
  WASH Deionized Water 3x 0:00 min.
8 Counterstain Refine Detection Kit Hematoxylin*  5 min. 
  WASH Deionized Water 0:00 min. 
  WASH BOND™ Wash Solution  0:00 min. 
  WASH Deionized Water 0:00 min. 
9 Dehydration (Offline):    
  Incubate sections in 95% ethanol two times for 10 seconds each.  
  Repeat in 100% ethanol, incubating sections two times for 10 seconds each.  
  Repeat in xylene, incubating sections two times for 10 seconds each.  
10 Mount sections with coverslips and #14177 SignalStain® Mounting Medium  
       
  Optional Custom wash:  BOND™ Wash Solution 2:00
    BOND™ Wash Solution Dispenser Type: OPEN 0:00
    BOND™ Wash Solution 2:00
    BOND™ Wash Solution Dispenser Type: OPEN 0:00
    BOND™ Wash Solution 0:00
    Deionized Water 0:00

*Reagent included in BOND™ Polymer Refine Detection Kit (Catalog No: DS9800)

LEICA® is a registered trademark of Leica Microsystems IR GmbH.

BOND™ is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.

posted August 2018

revised September 2018

Protocol Id: 1444

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X EDTA Unmasking Solution: To prepare 250 mL of 1X EDTA unmasking solution, dilute 25 ml of SignalStain® EDTA Unmasking Solution (10X) (#14747) with 225 mL of dH2O.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For EDTA: Heat slides in a microwave submersed in 1X EDTA unmasking solution until boiling is initiated; follow with 15 min at a sub-boiling temperature (95°-98°C). No cooling is necessary.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

DETECTION REAGENT/SUBSTRATE COMPATIBILITY
RECOMMENDED
DETECTION REAGENTS
SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653
COMPATIBLE
CHROMOGEN
SignalStain® DAB Substrate Kit #8059 SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632  

NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.


posted February 2010

revised June 2020

Protocol Id: 284

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Methanol, 100%
  4. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  5. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  6. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  7. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 1X PBS.
    NOTE: Formaldehyde is toxic, use only in fume hood.
  2. Allow cells to fix for 15 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Methanol Permeabilization Step: Cover cells with ice-cold 100% methanol (use enough to cover completely to a depth of 3–5 mm, DO NOT LET DRY), incubate in methanol for 10 minutes at –20°C, rinse in 1X PBS for 5 minutes.
  2. Block specimen in Blocking Buffer for 60 minutes.
  3. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
  4. Aspirate blocking solution, apply diluted primary antibody.
  5. Incubate overnight at 4°C.
  6. Rinse three times in 1X PBS for 5 minutes each.
  7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  8. Rinse in 1X PBS as in step 6.
  9. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  10. For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.

posted November 2006

revised December 2010

Protocol Id: 32

Flow Cytometry, Methanol Permeabilization Protocol for Rabbit Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  5. Recommended Anti-Rabbit secondary antibodies::
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #79408

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
  7. Incubate for 30 min at room temperature. Protect from light.
  8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  9. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised June 2020

Protocol Id: 404

Specific for product: SimpleChIP® Plus Sonication Chromatin IP Kit #56383.

Required Reagents

Reagents Included:

  1. Glycine Solution (10X) #7005
  2. ChIP Sonication Cell Lysis Buffer (2X) #96529
  3. ChIP Sonication Nuclear Lysis Buffer #28778
  4. ChIP Buffer (10X) #7008
  5. ChIP Elution Buffer (2X) #7009
  6. 5 M NaCl #7010
  7. ChIP-Grade Protein G Magnetic Beads #9006
  8. DNA Binding Buffer #10007
  9. DNA Wash Buffer (add 4x volume ethanol before use) #10008
  10. DNA Elution Buffer #10009
  11. DNA Purification Columns #10010
  12. Protease Inhibitor Cocktail (200X) #7012
  13. RNase A (10 mg/ml) #7013
  14. Proteinase K (20 mg/ml) #10012
  15. SimpleChIP® Human RPL30 Exon 3 Primers 1 #7014
  16. SimpleChIP® Mouse RPL30 Intron 2 Primers 1 #7015
  17. Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620
  18. Normal Rabbit IgG #2729

Reagents Not Included:

  1. Magnetic Separation Rack #7017 / #14654
  2. Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile) #9872
  3. Nuclease-free Water #12931
  4. SimpleChIP® Universal qPCR Master Mix #88989
  5. Ethanol (96-100%)
  6. Formaldehyde (37% Stock)
! This ! signifies an important step in the protocol regarding volume changes based on the number of immunoprecipitation preparations (IP preps). One IP prep is defined as 4 x 106 tissue cultured cells or 25 mg or disaggregated tissue.
!! This !! signifies an important step to dilute a buffer before proceeding.
SAFE STOP This is a safe stopping point in the protocol, if stopping is necessary.

I. Tissue Cross-linking and Sample Preparation

When harvesting tissue, remove unwanted material from the sample, such as fat and necrotic material. Tissue can be processed and cross-linked immediately or frozen on dry ice for processing later. For optimal ChIP results, use 25 mg of tissue for each immunoprecipitation to be performed. An additional 5 mg of tissue should be processed for Analysis of Chromatin Digestion and Concentration and for use as input chromatin (Section IV). The chromatin yield does vary between tissue types, and some tissues may require more than 25 mg for each immunoprecipitation.

One chromatin preparation is defined as 100 to 150 mg of tissue. This recommended amount of tissue accounts for potential low yield with some tissue types and also ensures efficient chromatin fragmentation during sonication. Please see Appendix A for more information regarding the expected chromatin yield for different types of tissue.

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of chromatin preps in the experiment.

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and Glycine Solution (10X) #7005. Make sure PIC is completely thawed.
  • Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 µl 200X PIC per chromatin preparation and place on ice.
  • Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml 2X ChIP Sonication Cell Lysis Buffer #96529 + 0.5 ml water) + 5 µl 200X PIC per chromatin preparation and place on ice.
  • Prepare 28 µl of 37% formaldehyde per chromatin preparation and keep at room temperature. Alternatively, 62.5 µl of 16% methanol-free formaldehyde can be used. Use fresh formaldehyde that is not past the manufacturer's expiration date.
  1. Weigh the fresh or frozen tissue sample. Use 100 to 150 mg of tissue per chromatin preparation.
  2. Place tissue sample in a petri dish and mince into 1-2 mm cubes using a clean scalpel or razor blade. Keep dish on ice. It is important to keep the tissue cold to avoid protein degradation.
  3. Transfer minced tissue to a 15 ml conical tube and add 1 ml of ice-cold PBS + PIC per chromatin preparation.
  4. To crosslink proteins to DNA, add 28 µl of 37% formaldehyde or 62.5 µl of 16% methanol-free formaldehyde per 1 ml of PBS + PIC and keep at room temperature for at least 10 min. Final formaldehyde concentration is 1%. For histone modification ChIP, 10 min fixation is sufficient; however, for transcription factor ChIP, we recommend fixation for 10 to 30 min, and for transcription cofactor ChIP, we recommend fixation for 30 min (see Figure 7 in Appendix B).
  5. Stop cross-linking by adding 100 µl of 10X Glycine #7005 per 1 ml of PBS + PIC. Mix and incubate on ice for 5 min.
  6. Centrifuge tissue at 1,200 x g for 5 min at 4°C.
  7. Remove supernatant and wash with 1 ml of ice-cold PBS + PIC per chromatin preparation.
  8. Centrifuge at 1,200 x g for 5 min at 4°C.
  9. Repeat step 7 and 8 one additional time.
  10. Remove supernatant and resuspend tissue in 1 ml 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation. (SAFE STOP) Alternatively, samples may be stored at -80°C before disaggregation for up to 3 months.
  11. Transfer tissue suspension to a Dounce homogenizer using a cut pipet tip.
  12. Use tight fitting pestle (Type A) to disaggregate tissue pieces with 20 strokes or until no chunks of tissue are observed.
  13. Transfer cell suspension to a 1.5 ml tube and immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III).

II. Cell Culture Cross-linking and Sample Preparation

For optimal ChIP results, use approximately 4 x 106 cells for each immunoprecipitation to be performed. For HCT 116 cells, this is equivalent to 1/3 of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. An additional 1 x 106 cells should be processed for Analysis of Chromatin Digestion and Concentration and for use as input chromatin (Section IV).

One chromatin preparation is defined as 1 x 107 to 2 x 107 cells. This recommended cell number accounts for potential low yield with some cell types and also ensures efficient chromatin fragmentation during sonication.

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of 15 cm tissue culture dishes (or 20 ml suspension cells) used in the experiment.

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and Glycine Solution(10X) #7005. Make sure PIC is completely thawed.
  • Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 µl 200X PIC per 15 cm dish (or 20 ml suspension cells) to be processed and place on ice.
  • Prepare 40 ml of PBS per 15 cm dish (or 20 ml suspension cells) to be processed and place on ice.
  • Prepare 540 µl of 37% formaldehyde per 15 cm dish of cells (or 20 ml suspension cells) to be processed and keep at room temperature. Alternatively, 1.25 ml of 16% methanol-free formaldehyde can be used. Use fresh formaldehyde that is not past the manufacturer's expiration date.
  • Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml 2X ChIP Sonication Cell Lysis Buffer #96529 + 0.5 ml water) + 5 µl 200X PIC per chromatin preparation.
  1. To crosslink proteins to DNA, add 540 µl of 37% formaldehyde or 1.25 ml of 16% methanol-free formaldehyde to each 15 cm culture dish containing 20 ml medium (or 20 ml suspension cells). For optimal fixation of suspension cells, cell density should be less than 0.5 x 106 cells/ml at fixation. Swirl briefly to mix and incubate 10 min at room temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde may result in a color change of the medium.
  2. Add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly to mix, and incubate 5 min at room temperature. Addition of glycine may result in a color change of the medium.
  3. For suspension cells:
    1. Transfer cells to a 50 ml conical tube, centrifuge 1,000 x g for 5 min at 4°C, and wash pellet two times with 20 ml ice-cold PBS. Remove PBS and proceed to step 3b. Alternatively, cell pellets can be frozen on dry ice and stored at -80°C for later use.
    2. Resuspend up to 2 x 107 cells per 1 ml of 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation, and immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III). (SAFE STOP) Alternatively, samples may be stored at -80°C for up to 3 months.
  4. For adherent cells:
    1. Remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from culture dish each time.
    2. Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into cold buffer. Combine cells from all culture dishes into one 15 ml conical tube.
    3. Centrifuge cells at 1,000 x g for 5 min at 4°C. Remove PBS and proceed to step 4d. Alternatively, cell pellets can be frozen on dry ice and stored at -80°C for later use
    4. Resuspend up to 2 x 107 cells per 1 ml of 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation and immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III). (SAFE STOP) Alternatively, samples may be stored at -80°C for up to 3 months.

III. Nuclei Preparation and Chromatin Fragmentation

One chromatin preparation is defined as 100 to 150 mg of tissue or 1 x 107to 2 x 107tissue culture cells. Multiple chromatin preparations can be performed simultaneously, as long as the amounts of buffers are scaled appropriately and sonication is performed on 1 ml samples. The number of cells and volume of sample used for sonication is critical for generation of appropriately sized chromatin fragments.

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of chromatin preps in the experiment.

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure it is completely thawed prior to use.
  • Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml 2X ChIP Sonication Cell Lysis Buffer #96529 + 0.5 ml water) + 5 µl 200X PIC per chromatin preparation.
  • Prepare 1 ml ChIP Sonication Nuclear Lysis Buffer #28778 + 5 µl 200X PIC per chromatin preparation.
  1. Incubate cell suspension in 1X ChIP Sonication Cell Lysis Buffer + PIC from Section I or Section II on ice for 10 min.
  2. Pellet cells at 5,000 x g for 5 min at 4°C. Remove supernatant and resuspend pellet a second time in 1 ml ice-cold 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation.
  3. Incubate cell suspension on ice for 5 min. Pellet cells at 5,000 x g for 5 min at 4°C. Please note that cross-linked cells may not be completely lysed until sonication.
  4. Resuspend cells in 1 ml ice-cold ChIP Sonication Nuclear Lysis Buffer #28778 + PIC per chromatin preparation and incubate on ice for 10 min. Transfer 1 ml of cell suspension to an appropriately sized tube for sonication. Please note that cross-linked cells and nuclei may not be completely lysed until sonication.
  5. Fragment chromatin by sonication. Sonication conditions may need to be determined empirically by testing different sonicator power settings and durations of sonication. Optimal sonication conditions will generate chromatin in which 60 to 90% of the chromatin fragments are smaller than 1kb. Longer cross-linking times may decrease the proportion of fragments being smaller than 1 kb to 30 to 60% (see Figure 7 in Appendix B and Figure 8 in Appendix C). Use the minimal number of sonication cycles required to generate the desired length of chromatin fragments, as over-sonication can result in reduction or loss of signal due to harsh treatment of the chromatin.
    • For each sonication sample, we recommend using 100-150 mg of tissue or 1 x 107to 2 x 107cells per 1 ml ChIP Sonication Nuclear Lysis Buffer. Sonicating in larger volumes and/or using greater concentration of cells will decrease the efficiency of chromatin fragmentation.
    • Using a Branson Digital Sonifier D250 probe sonicator with a 1/8-inch Micro Tip, 8 min of 1 sec on/1 sec off sonication cycle (4 min of sonication time) at 50% amplitude typically gives good fragmentation and chromatin IP efficiency.
    • Be sure to keep the chromatin sample cool during sonication by keeping the tube containing the chromatin in an ice water bath during and in between the sonication steps. Do not allow the probe to touch the bottom or wall of the tube. Stop sonication and adjust the position of tube if chromatin sample foams during sonication.
  6. Clarify lysates by centrifugation at 21,000 x g in a microcentrifuge for 10 min at 4°C.
  7. Transfer supernatant to a new tube. (SAFE STOP) This is the cross-linked chromatin preparation, which should be stored at -80°C until further use. Remove 50 µl of the chromatin preparation for Analysis of Chromatin Digestion and Concentration (Section IV).

IV. Analysis of Chromatin Fragmentation and Concentration (Recommended Step)

  1. To the 50 µl chromatin sample (from Step 7 in Section III), add 100 µl nuclease-free water, 6 µl 5 M NaCl #7010, and 2 µl RNAse A#7013. Vortex to mix and incubate samples at 37°C for 30 min.
  2. To each RNase A-digested sample, add 2 µl Proteinase K #10012. Vortex to mix and incubate samples at 65°C for 2 hr.
  3. Purify DNA from samples using DNA purification spin columns as described in Section VII. (SAFE STOP) DNA may be stored at -20°C for up to 6 months.
  4. After purification of DNA, remove a 10 µl sample and determine DNA fragment size by electrophoresis on a 1% agarose gel. A DNA smear ranging from 200 bp to several kb is expected (see Figure 7 in Appendix C). Approximately 60 to 90% of total DNA fragments should be less than 1 kb. Refer to Appendix B for optimization of sonication conditions.
  5. Determine DNA concentration using a spectrophotometer. Ideally, DNA concentration should be between 50 and 200 µg/ml.

V. Chromatin Immunoprecipitation

For optimal ChIP results, use approximately 5 to 10 µg of sonicated, cross-linked chromatin (as determined in Section IV) per immunoprecipitation. This should be roughly equivalent to a single 100 µl IP prep from 25 mg of disaggregated tissue or 4x106 tissue culture cells. Typically, 100 µl of digested chromatin is diluted into 400 µl 1X ChIP Buffer prior to the addition of antibodies. However, if more than 100 µl of chromatin is required per IP, the cross-linked chromatin preparation must be diluted into 1X ChIP buffer at a dilution ratio of 1:4. No additional protein G magnetic beads are necessary in this case, although prolonged incubation time with beads is helpful.

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of immunoprecipitations in the experiment.

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure PIC is completely thawed.
  • Remove and warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.
  • Thaw fragmented chromatin preparation (from Step 7 in Section III) and place on ice.
  • Prepare low salt wash: 3 ml 1X ChIP Buffer (300 µl 10X ChIP Buffer #7008 + 2.7 ml water) per immunoprecipitation. Keep on ice.
  • Prepare high salt wash: 1 ml 1X ChIP Buffer (100 µl 10X ChIP Buffer #7008 + 900 µl water) + 70 µl 5M NaCl #7010 per immunoprecipitation. Keep on ice.
  1. In one tube, prepare enough 1X ChIP Buffer + PIC for the dilution of sonicated chromatin into the desired number of immunoprecipitations. For each immunoprecipitation, dilute chromatin (5-10 µg of DNA) with 1X ChIP Buffer + PIC into a total of 500 µl. For efficient immunoprecipitation, chromatin must be diluted into 1 x ChIP buffer at a dilution factor of 1:4 or greater. Place mix on ice. When determining the number of immunoprecipitations, remember to include the positive control Histone H3 (D2B12) XP® Rabbit mAb#4620 and negative control Normal Rabbit IgG antibody #2729 samples.
  2. Remove a 10 µl sample of the diluted chromatin and transfer to a microfuge tube. This is your 2% Input Sample, which can be stored at -20°C until further use (Step 1 in Section VI).
  3. For each immunoprecipitation, aliquot 500 µl the diluted chromatin into a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody required per IP varies and should be used according to the manufacturers' recommendations. For the positive control Histone H3 (D2B12) XP® Rabbit mAb#4620, add 10 µl to the IP sample. For the negative control Normal Rabbit IgG #2729, add 1 µl (1 µg) to 2 µl (2 µg) to the IP sample. Incubate IP samples 4 hr to overnight at 4°C with rotation.
  4. NOTE: Most antibodies from Cell Signaling Technology work optimally between 1 and 2 µg per IP sample. In the case where there are multiple samples with varying concentrations, it is best to match the negative control Normal Rabbit IgG #2729 to the highest antibody concentration.

  5. Resuspend ChIP-Grade Protein G Magnetic Beads #9006 by gently vortexing. Immediately add 30 µl of Protein G Magnetic Beads to each IP reaction and incubate for 2 hr at 4°C with rotation. Alternatively, beads can be added to the overnight antibody incubation in Step 3, but this may increase background signal.
  6. Pellet Protein G Magnetic Beads in each immunoprecipitation by placing the tubes in a magnetic separation rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant.
  7. Wash Protein G Magnetic Beads by adding 1 ml of low salt wash to the beads and incubate at 4°C for 5 min with rotation. Repeat steps 5 and 6 two additional times for a total of 3 low salt washes.
  8. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
  9. Pellet Protein G Magnetic Beads in each immunoprecipitation by placing the tubes in a magnetic separation rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant. Immediately proceed to Section VI.

VI. Elution of Chromatin from Antibody/Protein G Magnetic Beads and Reversal of Cross-links

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of immunoprecipitations in the experiment.

  • Remove and warm 2X ChIP Elution Buffer #7009 in a 37°C water bath and ensure SDS is in solution.
  • Set a water bath or thermomixer to 65°C.
  • Prepare 150 µl 1X ChIP Elution Buffer (75 µl 2X ChIP Elution Buffer #7009 + 75 µl water) for each immunoprecipitation and the 2% input samples.
  1. Add 150 µl of the 1X ChIP Elution Buffer to the 2% input sample tubes and set aside at room temperature until Step 7.
  2. Add 150 µl 1X ChIP Elution Buffer to each IP sample.
  3. Elute chromatin from the antibody/Protein G Magnetic Beads for 30 min at 65°C with gentle vortexing (1,200 rpm). A thermomixer works best for this step; however a 65°C water bath with frequent mixing is also sufficient. Alternatively, elutions can be performed at room temperature with rotation, but may not be as complete.
  4. Briefly centrifuge samples at 10,000 x g for 10 sec to remove evaporated sample from the microcentrifuge tube cap.
  5. Pellet Protein G Magnetic Beads by placing the tubes in a magnetic separation rack and wait 1 to 2 min for solution to clear.
  6. Carefully transfer eluted chromatin supernatant to a new tube.
  7. To all tubes, including the 2% input samples from Step 1, reverse cross-links by adding 6 µl 5M NaCl #7010 and 2 µl Proteinase K #10012, and incubate 2 hr at 65°C. This incubation can be extended overnight.
  8. Immediately proceed to Section VII. (SAFE STOP) Alternatively, samples can be stored at -20°C for up to 4 days. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Buffer #10007 (Section VII, Step 1).

VII. DNA Purification Using Spin Columns:

Before starting:

  • (!!) Add 24 ml of ethanol (96-100%) to DNA Wash Buffer #10008 before use. This step only has to be performed once prior to the first set of DNA purifications.
  • Prepare one DNA purification spin column and collection tube #10010 for each DNA sample from Section VI.
  1. Add 750 µl DNA Binding Buffer #10007 to each DNA sample and vortex briefly.
    • 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.
  2. Transfer 450 µl of each DNA sample from Step 1 to a DNA spin column in collection tube.
  3. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  4. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  5. Transfer the remaining 450 µl of each DNA sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
  6. Add 750 µl of DNA Wash Buffer #10008 to the spin column in collection tube.
  7. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  8. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  9. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec
  10. Discard collection tube and liquid. Retain spin column.
  11. Add 50 µl of DNA Elution Buffer #10009 to each spin column and place into a clean 1.5 ml microcentrifuge tube.
  12. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec to elute DNA.
  13. Remove and discard DNA spin column. Eluate is now purified DNA. (SAFE STOP) Samples can be stored at -20°C for up to 6 months.

VIII. Quantification of DNA by PCR:

Recommendations:

  • Use filtered pipette tips to minimize risk of contamination.
  • The control primers included in the kit are specific for the human or mouse RPL30 gene (#7014 or #7015) and can be used for either standard PCR or quantitative real-time PCR. If the user is performing ChIP from another species, it is recommended that the user design the appropriate specific primers to DNA and determine the optimal PCR conditions.
  • A Hot-Start Taq polymerase is recommended to minimize the risk of nonspecific PCR products.
  • PCR primer selection is critical. Primers should be designed with close adherence to the following criteria:
    Primer length: 24 nucleotides
    Optimum Tm: 60°C
    Optimum GC: 50%
    Amplicon size: 150 to 200 bp (for standard PCR)
      80 to 160 bp (for real-time quantitative PCR)

Standard PCR Method:

  1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. These should include the 2% input sample, the positive control histone H3 sample, the negative control normal rabbit IgG sample, and a tube with no DNA to control for DNA contamination.
  2. Add 2 µl of the appropriate DNA sample to each tube.
  3. Prepare a master reaction mix as described below, making sure to add enough reagent for two extra tubes to account for loss of volume. Add 18 µl of master mix to each reaction tube.
    Reagent Volume for 1 PCR Reaction (18 µl)
    Nuclease-free H2O 12.5 µl
    10X PCR Buffer 2.0 µl
    4 mM dNTP Mix 1.0 µl
    5 µM RPL30 Primers 2.0 µl
    Taq DNA Polymerase 0.5 µl
  4. Start the following PCR reaction program:
    1. Initial Denaturation 95°C 5 min
    2. Denature 95°C 30 sec
    3. Anneal 62°C 30 sec
    4. Extension 72°C 30 sec
    5. Repeat Steps b-d for a total of 34 cycles.
    6. Final Extension 72°C 5 min
  5. Remove 10 µl of each PCR product for analysis by 2% agarose gel or 10% polyacrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR product is 161 bp for human RPL30 #7014 and 159 bp for mouse RPL30 #7015.

Real-Time Quantitative PCR Method:

  1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. PCR reactions should include the positive control histone H3 sample, the negative control normal rabbit IgG sample, a tube with no DNA to control for contamination, and a serial dilution of the 2% input chromatin DNA (undiluted, 1:5, 1:25, 1:125) to create a standard curve and determine the efficiency of amplification.
  2. Add 2 µl of the appropriate DNA sample to each tube or well of the PCR plate.
  3. Prepare a master reaction mix as described below. Setup 2-3 replicates for each PCR reaction. Add enough reagents to account for loss of volume. Add 18 µl of reaction mix to each PCR reaction tube or well.(SAFE STOP) If necessary cover plate with aluminum foil to avoid light and store at 4°C up to 4 hours or -20°C overnight until machine is ready for use.
    Reagent Volume for 1 PCR Reaction (18 µl)
    Nuclease-free H2O 6 µl
    5 µM RPL30 Primers 2 µl
    SimpleChIP® Universal qPCR Master Mix #88989 10 µl
  4. Start the following PCR reaction program:
    a. Initial Denaturation 95°C 3 min
    b. Denature 95°C 15 sec
    c. Anneal and Extension: 60°C 60 sec
    d. Repeat steps b and c for a total of 40 cycles.  
  5. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.

    Percent Input = 2% x 2(C[T] 2%Input Sample - C[T] IP Sample)
    C[T] = CT = Average threshold cycle of PCR reaction

IX. NG-Sequencing Library Construction

The immuno-enriched DNA samples prepared with this kit are directly compatible with ChIP-seq. For downstream NG-sequencing DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina® platforms, we recommend SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 and its associated index primers SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers) #29580 or SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538.

Recommendations:

  • For transcription factor or co-factor ChIP-seq, use at least 5 ng of ChIP-enriched DNA and amplification of the adaptor-ligated DNA with 10 cycles of PCR.
  • For total histone and histone modifications, or input samples, start with 50 ng of ChIP-enriched DNA and amplification of the adaptor-ligated DNA with 6 cycles of PCR.
  • For library construction of ChIP-enriched DNA for all target types, perform cleanup of adaptor-ligated DNA without size selection.
  • After DNA library construction, check the DNA library for presence of adaptor dimers (~140 bp) using an Agilent High Sensitivity DNA Kit (Agilent Technologies, Cat# G2938-90322), or by agarose gel electrophoresis with 50-100 ng DNA on a 2% agarose TAE gel. If adaptor dimers are present, repeat cleanup of PCR amplified material.
  • The quality of the library can also be confirmed using qPCR and primer sets to known positive and negative target loci. Positive primer pairs should still give the same high signal compared to negative primers as seen in the original qPCR analysis of ChIP-enriched DNA.
  • After final cleanup and quality checks, prepare final purified library samples at 2-10 nM for high throughput sequencing.

Appendix A: Expected Chromatin Yield

When harvesting cross-linked chromatin from tissue samples, the yield of chromatin can vary significantly between tissue types. The table to the right provides a range for the expected yield of chromatin from 100 mg of tissue compared to 2 x 107 HCT 116 cells, and the expected DNA concentration, as determined in Section IV of the protocol. For optimal ChIP results, we recommend using 5 to 10 µg of sonicated, cross-linked chromatin per immunoprecipitation.

Tissue/Cell Total Chromatin Yield Expected DNA Concentration
Liver 50 µg per 100 mg tissue 150 µg/ml
Brain 25 µg per 100 mg tissue 50 µg/ml
Heart 105 µg per 100 mg tissue 20 µg/ml
HCT 116 100-150 µg per 2 x 107 cells 100-150 µg/ml

Appendix B: Optimization of Formaldehyde Fixation

Transcription factors and cofactors bind to chromatin DNA more loosely than histone proteins. As a result, they tend to dissociate from chromatin during sonication. Additional fixation time can result in increased capture of transcription factors and cofactors in the ChIP assay, especially with tissue samples. As shown in Figure 7, increasing the fixation time from 10 min to 30 min may reduce chromatin fragmentation size (left panel), but it significantly enhances enrichment of both cofactors RING1B and SUZ12 in heart tissue, as indicated by ChIP-qPCR (middle and right panels).

Typically, 10 min fixation is sufficient for histone modification ChIP with both cell and tissue samples, whereas transcription factors and cofactors may require additional fixation up to 30 min, especially with tissue samples.

FIGURE 7. Mouse heart (H), brain (B), and liver (L) were cross-linked for 10 min or 30 min.

FIGURE 7. Mouse heart (H), brain (B), and liver (L) were cross-linked for 10 min or 30 min, as indicated (left panel). The chromatin was prepared and sonicated, DNA was purified and separated by electrophoresis on a 1% agarose gel. In the ChIP-qPCR assay (middle and right panels), chromatin immunoprecipitations were performed with either RING1B (D22F2) XP® Rabbit mAb #5694 or SUZ12 (D39F6) XP® Rabbit mAb #3737. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse HoxD10 Exon 1 Primers #7429, SimpleChIP® Mouse HoxA1 Promoter Primers #7341, and SimpleChIP® Mouse GAPDH Intron 2 Primers #8986. The amount of immunoprecipitated DNA in each sample is represented as normalized signal to the negative GAPDH locus (equivalent to one).

Appendix C: Optimization of Chromatin Fragmentation

Optimal conditions for the fragmentation of cross-linked chromatin DNA is highly dependent on the number of cells, volume of sample, length of sonication, and sonicator power setting used. For each sonication sample, we recommend using 100-150 mg of tissue or 1 x 107 to 2 x 107 cells per 1 ml ChIP Sonication Nuclear Lysis Buffer. Below is a protocol for determination of the optimal sonication conditions for a specific tissue or cell type.

  1. Prepare cross-linked nuclei from 100 to 150 mg of tissue or 1 x 107 to 2 x 107 cells, as described in Sections I, II, and III. Stop after Step 4 of Section III and proceed as described below.
  2. Fragment chromatin by sonication. Optimal sonication conditions can be determined for a given sonicator by varying the number of rounds or duration of sonication at a given power setting (see Step 5 in Section III for optimal power setting using a Branson Digital Sonifier 250 probe sonicator). To determine optimal sonication conditions, set up a sonication time-course experiment and remove 50 µl samples of chromatin after a given round or duration of sonication. For example, take chromatin samples after each 1 to 2 min of sonication.
  3. Clarify chromatin samples by centrifugation at 21,000 x g in a microcentrifuge for 10 min at 4°C.
  4. Transfer supernatants to new microfuge tubes and add 100 µl nuclease-free water, 6 µl 5 M NaCl #7010, and 2 µl RNAse A #7013. Vortex to mix and incubate samples at 37°C for 30 min.
  5. To each RNase A-digested sample, add 2 µl Proteinase K #10012. Vortex to mix and incubate samples at 65°C for 2 hr.
  6. Remove 20 µl of each sample and determine DNA fragment size
  7. Choose the sonication conditions that generate optimal DNA fragment size and use for chromatin preparation in Step 5 of Section III. If optimal sonication conditions are not achieved, increase or decrease the power setting of the sonicator and repeat the sonication time course.

NOTE: Optimal sonication conditions can vary with different sample types and fixation times. Use the minimal number of sonication cycles required to generate the desired length of chromatin fragments. Over sonication, as indicated by more than 80% of total DNA fragments less than 500 bp, can result in excessive damage to the chromatin and result in lower immunoprecipitation efficiency (see Figure 8, right panel).

  • For sonication of cells fixed for 10 min, optimal sonication conditions will generate a DNA smear with approximately 90% of total DNA fragments less than 1 kb (see Figure 8, left panel). Increasing the fixation time to 30 min will limit fragmentation, generating a DNA smear with approximately 60% of total DNA fragments less than 1 kb.
  • For sonication of tissues fixed for 10 min, optimal sonication conditions will generate a DNA smear with approximately 60% of total DNA fragments less than 1 kb. Increasing the fixation time to 30 min will limit fragmentation, generating a DNA smear with approximately 30% of total DNA fragments less than 1 kb (see Figure 7, left panel).
FIGURE 8. Chromatin immunoprecipitations were performed with 2 x 10^7 HCT 116 cells

FIGURE 8. HCT 116 cells were cross-linked for 10 min and sonicated for the time indicated (left panel). DNA was purified as described in Section IV of the SimpleChIP® Plus Sonication Chromatin IP Kit #56383, and 20 µl of purified DNA was separated by electrophoresis on a 1% agarose gel. As shown in the left panel, increasing cycles of sonication reduces the size of chromatin fragments. Chromatin immunoprecipitations were performed with either Non-phospho (Active) Β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb #8814, TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, or Normal Rabbit IgG #2729 using SimpleChIP® Plus Sonication Chromatin IP Kit #56383. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CaMK2D Intron 3 Primers #5111 and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 100%; right panel). As shown, 4 min of chromatin sonication is optimal when using a Branson Digital Sonifier D250 probe sonicator with a 1/8 inch Micro Tip. Over-sonication significantly impairs enrichment of both cofactor beta-catenin and transcription factor TCF4/TCF7L2 containing chromatin.

Appendix D: Troubleshooting Guide

Shorten the crosslinking time within 10-30 minute range. Reduce the amount of cell/tissues per sonication. Conduct sonication time course.
Problem Possible Causes Recommendation
1. Concentration of the fragmented chromatin is too low.

Cell/nuclear lysis is incomplete.

Not enough cells were used for the chromatin preparation.

If DNA concentration of the chromatin preparation is close to 50 µg/ml, add additional chromatin to each IP to give at least 5 µg/IP and continue with protocol.

Count a separate plate of cells before cross-linking to determine an accurate cell number.

2. Chromatin is under-fragmented and fragments are too large (>50% above 1.5kb).

Cells may have been over cross-linked.

Too many cells/tissues were processed.

Shorten the crosslinking time within 10-30 minute range. Reduce the amount of cell/tissues per sonication. Conduct sonication time course.

3. Chromatin is over-fragmented (>90% under 300 bp).

Sonication condition is too harsh.

Conduct a sonication time course to find a minimum output/duration to achieve appropriate sonication.

4. No product or very little product in the input PCR reactions.

Not enough DNA added to the PCR reaction or conditions are not optimal.

PCR amplified region may span nucleosome-free region.

Not enough chromatin added to the IP or chromatin is over-sonicated

Add more DNA to the PCR reaction or increase the number of amplification cycles.

Optimize the PCR conditions for experimental primer set using purified DNA from cross-linked and sonicated chromatin. For optimal ChIP results add 5-10 µg chromatin per IP. See recommendations for problems 1 and 3 above.

5. No product in the positive control Histone H3-IP RPL30 PCR reaction.

Not enough chromatin or antibody added to the IP reaction or IP incubation time is too short.

Incomplete elution of chromatin from Protein G beads.

Be sure to add 5-10 µg of chromatin and 10 µl of antibody to each IP reaction and incubate with antibody over-night and an additional 2 hr after adding Protein G beads.

Elution of chromatin from Protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.

6. Quantity of product in the negative control Rabbit IgG-IP and positive control Histone H3-IP PCR reactions is equivalent.

Too much or not enough chromatin added to the IP reaction. Alternatively, too much antibody added to the IP reaction.

Too much DNA added to the PCR reaction or too many cycles of amplification.

Add no more than 15 µg of chromatin and 10 µl of histone H3 antibody to each IP reaction. Reduce the amount of normal rabbit IgG to 1 µl per IP.

Add less DNA to the PCR reaction or decrease the number of PCR cycles. It is very important that the PCR products are analyzed within the linear amplification phase of PCR. Otherwise, the differences in quantities of starting DNA cannot be accurately measured.

7. No product in the Experimental Antibody-IP PCR reaction.

Not enough DNA added to the PCR reaction.

Not enough antibody added to the IP reaction.

Antibody does not work for IP.

Add more DNA to the PCR reaction or increase the number of amplification cycles.

Typically a range of 1 to 5 µg of antibody is added to the IP reaction; however, the exact amount depends greatly on the individual antibody.

Increase the amount of antibody added to the IP. Find an alternate antibody source.

posted March 2017

revised June 2018

Protocol Id: 1384

Sonication Chromatin Immunoprecipitation Protocol

Specific for product: SimpleChIP® Plus Sonication Chromatin IP Kit #56383.

I. Tissue Cross-linking and Sample Preparation

When harvesting tissue, remove unwanted material from the sample, such as fat and necrotic material. Tissue can be processed and cross-linked immediately or frozen on dry ice for processing later. For optimal ChIP results, use 25 mg of tissue for each immunoprecipitation to be performed. An additional 5 mg of tissue should be processed for Analysis of Chromatin Digestion and Concentration and for use as input chromatin (Section IV). The chromatin yield does vary between tissue types, and some tissues may require more than 25 mg for each immunoprecipitation.

One chromatin preparation is defined as 100 to 150 mg of tissue. This recommended amount of tissue accounts for potential low yield with some tissue types and also ensures efficient chromatin fragmentation during sonication. Please see Appendix A for more information regarding the expected chromatin yield for different types of tissue.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and Glycine Solution (10X) #7005. Make sure PIC is completely thawed.
  • Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 µl 200X PIC per chromatin preparation and place on ice.
  • Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml 2X ChIP Sonication Cell Lysis Buffer #96529 + 0.5 ml water) + 5 µl 200X PIC per chromatin preparation and place on ice.
  • Prepare 28 µl of 37% formaldehyde per chromatin preparation and keep at room temperature. Alternatively, 62.5 µl of 16% methanol-free formaldehyde can be used. Use fresh formaldehyde that is not past the manufacturer's expiration date.
  1. Weigh the fresh or frozen tissue sample. Use 100 to 150 mg of tissue per chromatin preparation.
  2. Place tissue sample in a petri dish and mince into 1-2 mm cubes using a clean scalpel or razor blade. Keep dish on ice. It is important to keep the tissue cold to avoid protein degradation.
  3. Transfer minced tissue to a 15 ml conical tube and add 1 ml of ice-cold PBS + PIC per chromatin preparation.
  4. To crosslink proteins to DNA, add 28 µl of 37% formaldehyde or 62.5 µl of 16% methanol-free formaldehyde per 1 ml of PBS + PIC and keep at room temperature for at least 10 min. Final formaldehyde concentration is 1%. For histone modification ChIP, 10 min fixation is sufficient; however, for transcription factor ChIP, we recommend fixation for 10 to 30 min, and for transcription cofactor ChIP, we recommend fixation for 30 min (see Figure 7 in Appendix B).
  5. Stop cross-linking by adding 100 µl of 10X Glycine #7005 per 1 ml of PBS + PIC. Mix and incubate on ice for 5 min.
  6. Centrifuge tissue at 1,200 x g for 5 min at 4°C.
  7. Remove supernatant and wash with 1 ml of ice-cold PBS + PIC per chromatin preparation.
  8. Centrifuge at 1,200 x g for 5 min at 4°C.
  9. Repeat step 7 and 8 one additional time.
  10. Remove supernatant and resuspend tissue in 1 ml 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation.
  11. Transfer tissue suspension to a Dounce homogenizer using a cut pipet tip.
  12. Use tight fitting pestle (Type A) to disaggregate tissue pieces with 20 strokes or until no chunks of tissue are observed.
  13. Transfer cell suspension to a 1.5 ml tube and immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III).

II. Cell Culture Cross-linking and Sample Preparation

For optimal ChIP results, use approximately 4 x 106 cells for each immunoprecipitation to be performed. For HCT 116 cells, this is equivalent to 1/3 of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. An additional 1 x 106 cells should be processed for Analysis of Chromatin Digestion and Concentration and for use as input chromatin (Section IV).

One chromatin preparation is defined as 1 x 107 to 2 x 107 cells. This recommended cell number accounts for potential low yield with some cell types and also ensures efficient chromatin fragmentation during sonication.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and Glycine Solution(10X) #7005. Make sure PIC is completely thawed.
  • Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 µl 200X PIC per 15 cm dish to be processed and place on ice.
  • Prepare 40 ml of PBS per 15 cm dish to be processed and place on ice.
  • Prepare 540 µl of 37% formaldehyde per 15 cm dish of cells to be processed and keep at room temperature. Alternatively, 1.25 ml of 16% methanol-free formaldehyde can be used. Use fresh formaldehyde that is not past the manufacturer's expiration date.
  • Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml 2X ChIP Sonication Cell Lysis Buffer #96529 + 0.5 ml water) + 5 µl 200X PIC per chromatin preparation.
  1. To crosslink proteins to DNA, add 540 µl of 37% formaldehyde or 1.25 ml of 16% methanol-free formaldehyde to each 15 cm culture dish containing 20 ml medium. Swirl briefly to mix and incubate 10 min at room temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde may result in a color change of the medium.
  2. Add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly to mix, and incubate 5 min at room temperature. Addition of glycine may result in a color change of the medium.
  3. For suspension cells:
    1. Transfer cells to a 50 ml conical tube, centrifuge 1,000 x g for 5 min at 4°C, and wash pellet two times with 20 ml ice-cold PBS. Remove PBS and proceed to step 3b. Alternatively, cell pellets can be frozen on dry ice and stored at -80°C for later use.
    2. Resuspend up to 2 x 107 cells per 1 ml of 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation, and immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III).
  4. For adherent cells:
    1. Remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from culture dish each time.
    2. Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into cold buffer. Combine cells from all culture dishes into one 15 ml conical tube.
    3. Centrifuge cells at 1,000 x g for 5 min at 4°C. Remove PBS and proceed to step 4d. Alternatively, cell pellets can be frozen on dry ice and stored at -80°C for later use
    4. Resuspend up to 2 x 107 cells per 1 ml of 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation and immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III).

III. Nuclei Preparation and Chromatin Fragmentation

One chromatin preparation is defined as 100 to 150 mg of tissue or 1 x 107to 2 x 107tissue culture cells. Multiple chromatin preparations can be performed simultaneously, as long as the amounts of buffers are scaled appropriately and sonication is performed on 1 ml samples. The number of cells and volume of sample used for sonication is critical for generation of appropriately sized chromatin fragments.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure it is completely thawed prior to use.
  • Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml 2X ChIP Sonication Cell Lysis Buffer #96529 + 0.5 ml water) + 5 µl 200X PIC per chromatin preparation.
  • Prepare 1 ml ChIP Sonication Nuclear Lysis Buffer #28778 + 5 µl 200X PIC per chromatin preparation.
  1. Incubate cell suspension in 1X ChIP Sonication Cell Lysis Buffer + PIC from Section I or Section II on ice for 10 min.
  2. Pellet cells at 5,000 x g for 5 min at 4°C. Remove supernatant and resuspend pellet a second time in 1 ml ice-cold 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation.
  3. Incubate cell suspension on ice for 5 min. Pellet cells at 5,000 x g for 5 min at 4°C. Please note that cross-linked cells may not be completely lysed until sonication.
  4. Resuspend cells in 1 ml ice-cold ChIP Sonication Nuclear Lysis Buffer #28778 + PIC per chromatin preparation and incubate on ice for 10 min. Transfer 1 ml of cell suspension to an appropriately sized tube for sonication. Please note that cross-linked cells and nuclei may not be completely lysed until sonication.
  5. Fragment chromatin by sonication. Sonication conditions may need to be determined empirically by testing different sonicator power settings and durations of sonication. Optimal sonication conditions will generate chromatin in which 60 to 90% of the chromatin fragments are smaller than 1kb. Longer cross-linking times may decrease the proportion of fragments being smaller than 1 kb to 30 to 60% (see Figure 7 in Appendix B and Figure 8 in Appendix C). Use the minimal number of sonication cycles required to generate the desired length of chromatin fragments, as over-sonication can result in reduction or loss of signal due to harsh treatment of the chromatin.
    • For each sonication sample, we recommend using 100-150 mg of tissue or 1 x 107to 2 x 107cells per 1 ml ChIP Sonication Nuclear Lysis Buffer. Sonicating in larger volumes and/or using greater concentration of cells will decrease the efficiency of chromatin fragmentation.
    • Using a Branson Digital Sonifier D250 probe sonicator with a 1/8-inch Micro Tip, 8 min of 1 sec on/1 sec off sonication cycle (4 min of sonication time) at 50% amplitude typically gives good fragmentation and chromatin IP efficiency.
    • Be sure to keep the chromatin sample cool during sonication by keeping the tube containing the chromatin in an ice water bath during and in between the sonication steps. Do not allow the probe to touch the bottom or wall of the tube. Stop sonication and adjust the position of tube if chromatin sample foams during sonication.
  6. Clarify lysates by centrifugation at 21,000 x g in a microcentrifuge for 10 min at 4°C.
  7. Transfer supernatant to a new tube. This is the cross-linked chromatin preparation, which can be used immediately for immunoprecipitation, or stored at -80°C until further use. Remove 50 µl of the chromatin preparation for Analysis of Chromatin Digestion and Concentration (Section IV).

IV. Analysis of Chromatin Fragmentation and Concentration (Recommended Step)

  1. To the 50 µl chromatin sample (from Step 7 in Section III), add 100 µl nuclease-free water, 6 µl 5 M NaCl #7010, and 2 µl RNAse A#7013. Vortex to mix and incubate samples at 37°C for 30 min.
  2. To each RNase A-digested sample, add 2 µl Proteinase K #10012. Vortex to mix and incubate samples at 65°C for 2 hr.
  3. Purify DNA from samples using DNA purification spin columns as described in Section VII.
  4. After purification of DNA, remove a 10 µl sample and determine DNA fragment size by electrophoresis on a 1% agarose gel. A DNA smear ranging from 200 bp to several kb is expected (see Figure 7 in Appendix C). Approximately 60 to 90% of total DNA fragments should be less than 1 kb. Refer to Appendix B for optimization of sonication conditions.
  5. Determine DNA concentration using a spectrophotometer. Ideally, DNA concentration should be between 50 and 200 µg/ml.

V. Chromatin Immunoprecipitation

For optimal ChIP results, use approximately 5 to 10 µg of sonicated, cross-linked chromatin (as determined in Section IV) per immunoprecipitation. This should be roughly equivalent to a single 100 µl IP prep from 25 mg of disaggregated tissue or 4x106 tissue culture cells. Typically, 100 µl of digested chromatin is diluted into 400 µl 1X ChIP Buffer prior to the addition of antibodies. However, if more than 100 µl of chromatin is required per IP, the cross-linked chromatin preparation must be diluted into 1X ChIP buffer at a dilution ratio of 1:4. No additional protein G magnetic beads are necessary in this case, although prolonged incubation time with beads is helpful.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure PIC is completely thawed.
  • Remove and warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.
  • Thaw fragmented chromatin preparation (from Step 7 in Section III) and place on ice.
  • Prepare low salt wash: 3 ml 1X ChIP Buffer (300 µl 10X ChIP Buffer #7008 + 2.7 ml water) per immunoprecipitation. Keep on ice.
  • Prepare high salt wash: 1 ml 1X ChIP Buffer (100 µl 10X ChIP Buffer #7008 + 900 µl water) + 70 µl 5M NaCl #7010 per immunoprecipitation. Keep on ice.
  1. In one tube, prepare enough 1X ChIP Buffer + PIC for the dilution of sonicated chromatin into the desired number of immunoprecipitations. For each immunoprecipitation, dilute chromatin (5-10 µg of DNA) with 1X ChIP Buffer + PIC into a total of 500 µl. For efficient immunoprecipitation, chromatin must be diluted into 1 x ChIP buffer at a dilution factor of 1:4 or greater. Place mix on ice. When determining the number of immunoprecipitations, remember to include the positive control Histone H3 (D2B12) XP® Rabbit mAb#4620 and negative control Normal Rabbit IgG antibody #2729 samples.
  2. Remove a 10 µl sample of the diluted chromatin and transfer to a microfuge tube. This is your 2% Input Sample, which can be stored at -20°C until further use (Step 1 in Section VI).
  3. For each immunoprecipitation, aliquot 500 µl the diluted chromatin into a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody required per IP varies and should be used according to the manufacturers' recommendations. For the positive control Histone H3 (D2B12) XP® Rabbit mAb#4620, add 10 µl to the IP sample. For the negative control Normal Rabbit IgG #2729, add 1 µl (1 µg) to 2 µl (2 µg) to the IP sample. Incubate IP samples 4 hr to overnight at 4°C with rotation.
  4. NOTE: Most antibodies from Cell Signaling Technology work optimally between 1 and 2 µg per IP sample. In the case where there are multiple samples with varying concentrations, it is best to match the negative control Normal Rabbit IgG #2729 to the highest antibody concentration.

  5. Resuspend ChIP-Grade Protein G Magnetic Beads #9006 by gently vortexing. Immediately add 30 µl of Protein G Magnetic Beads to each IP reaction and incubate for 2 hr at 4°C with rotation. Alternatively, beads can be added to the overnight antibody incubation in Step 3, but this may increase background signal.
  6. Pellet Protein G Magnetic Beads in each immunoprecipitation by placing the tubes in a magnetic separation rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant.
  7. Wash Protein G Magnetic Beads by adding 1 ml of low salt wash to the beads and incubate at 4°C for 5 min with rotation. Repeat steps 6 and 7 two additional times for a total of 3 low salt washes.
  8. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
  9. Pellet Protein G Magnetic Beads in each immunoprecipitation by placing the tubes in a magnetic separation rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant. Immediately proceed to Section VI.

VI. Elution of Chromatin from Antibody/Protein G Magnetic Beads and Reversal of Cross-links

Before starting:

  • Remove and warm 2X ChIP Elution Buffer #7009 in a 37°C water bath and ensure SDS is in solution.
  • Set a water bath or thermomixer to 65°C.
  • Prepare 150 µl 1X ChIP Elution Buffer (75 µl 2X ChIP Elution Buffer #7009 + 75 µl water) for each immunoprecipitation and the 2% input samples.
  1. Add 150 µl of the 1X ChIP Elution Buffer to the 2% input sample tubes and set aside at room temperature until Step 7.
  2. Add 150 µl 1X ChIP Elution Buffer to each IP sample.
  3. Elute chromatin from the antibody/Protein G Magnetic Beads for 30 min at 65°C with gentle vortexing (1,200 rpm). A thermomixer works best for this step; however a 65°C water bath with frequent mixing is also sufficient. Alternatively, elutions can be performed at room temperature with rotation, but may not be as complete.
  4. Briefly centrifuge samples at 10,000 x g for 10 sec to remove evaporated sample from the microcentrifuge tube cap.
  5. Pellet Protein G Magnetic Beads by placing the tubes in a magnetic separation rack and wait 1 to 2 min for solution to clear.
  6. Carefully transfer eluted chromatin supernatant to a new tube.
  7. To all tubes, including the 2% input samples from Step 1, reverse cross-links by adding 6 µl 5M NaCl #7010 and 2 µl Proteinase K #10012, and incubate 2 hr at 65°C. This incubation can be extended overnight.
  8. Immediately proceed to Section VII. Alternatively, samples can be stored at -20°C. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Buffer #10007 (Section VII, Step 1).

VII. DNA Purification Using Spin Columns:

Before starting:

  • Add 24 ml of ethanol (96-100%) to DNA Wash Buffer #10008 before use. This step only has to be performed once prior to the first set of DNA purifications.
  • Prepare one DNA purification spin column and collection tube #10010 for each DNA sample from Section VI.
  1. Add 750 µl DNA Binding Buffer #10007 to each DNA sample and vortex briefly.
    • 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.
  2. Transfer 450 µl of each DNA sample from Step 1 to a DNA spin column in collection tube.
  3. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  4. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  5. Transfer the remaining 450 µl of each DNA sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
  6. Add 750 µl of DNA Wash Buffer #10008 to the spin column in collection tube.
  7. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  8. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  9. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec
  10. Discard collection tube and liquid. Retain spin column.
  11. Add 50 µl of DNA Elution Buffer #10009 to each spin column and place into a clean 1.5 ml microcentrifuge tube.
  12. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec to elute DNA.
  13. Remove and discard DNA spin column. Eluate is now purified DNA. Samples can be stored at -20°C.

VIII. Quantification of DNA by PCR:

Recommendations:

  • Use filtered pipette tips to minimize risk of contamination.
  • The control primers included in the kit are specific for the human or mouse RPL30 gene (#7014 or #7015) and can be used for either standard PCR or quantitative real-time PCR. If the user is performing ChIP from another species, it is recommended that the user design the appropriate specific primers to DNA and determine the optimal PCR conditions.
  • A Hot-Start Taq polymerase is recommended to minimize the risk of nonspecific PCR products.
  • PCR primer selection is critical. Primers should be designed with close adherence to the following criteria:
    Primer length: 24 nucleotides
    Optimum Tm: 60°C
    Optimum GC: 50%
    Amplicon size: 150 to 200 bp (for standard PCR)
    80 to 160 bp (for real-time quantitative PCR)

Standard PCR Method:

  1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. These should include the 2% input sample, the positive control histone H3 sample, the negative control normal rabbit IgG sample, and a tube with no DNA to control for DNA contamination.
  2. Add 2 µl of the appropriate DNA sample to each tube.
  3. Prepare a master reaction mix as described below, making sure to add enough reagent for two extra tubes to account for loss of volume. Add 18 µl of master mix to each reaction tube.
    Reagent Volume for 1 PCR Reaction (18 µl)
    Nuclease-free H2O 12.5 µl
    10X PCR Buffer 2.0 µl
    4 mM dNTP Mix 1.0 µl
    5 µM RPL30 Primers 2.0 µl
    Taq DNA Polymerase 0.5 µl
  4. Start the following PCR reaction program:
    1. Initial Denaturation 95°C 5 min
    2. Denature 95°C 30 sec
    3. Anneal 62°C 30 sec
    4. Extension 72°C 30 sec
    5. Repeat Steps b-d for a total of 34 cycles.
    6. Final Extension 72°C 5 min
  5. Remove 10 µl of each PCR product for analysis by 2% agarose gel or 10% polyacrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR product is 161 bp for human RPL30 #7014 and 159 bp for mouse RPL30 #7015.

Real-Time Quantitative PCR Method:

  1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. PCR reactions should include the positive control histone H3 sample, the negative control normal rabbit IgG sample, a tube with no DNA to control for contamination, and a serial dilution of the 2% input chromatin DNA (undiluted, 1:5, 1:25, 1:125) to create a standard curve and determine the efficiency of amplification.
  2. Add 2 µl of the appropriate DNA sample to each tube or well of the PCR plate.
  3. Prepare a master reaction mix as described below. Setup 2-3 replicates for each PCR reaction. Add enough reagents to account for loss of volume. Add 18 µl of reaction mix to each PCR reaction tube or well. Reagent Volume for 1 PCR Reaction (18 µl)
    Nuclease-free H2O 6 µl
    5 µM RPL30 Primers 2 µl
    SYBR-Green Reaction Mix 10 µl
  4. Start the following PCR reaction program:
    a. Initial Denaturation 95°C 3 min
    b. Denature 95°C 15 sec
    c. Anneal and Extension: 60°C 60 sec
    d. Repeat steps b and c for a total of 40 cycles.
  5. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.

    Percent Input = 2% x 2(C[T] 2%Input Sample - C[T] IP Sample)
    C[T] = CT = Average threshold cycle of PCR reaction

IX. NG-Sequencing Library Construction

The immuno-enriched DNA samples prepared with this kit are directly compatible with ChIP-seq. For downstream NG-sequencing DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina® platforms, we recommend NEBNext® Ultra™ II Library Prep Kit for Illumina® (New England Biolabs; Cat #E7645S/L).

Recommendations:

  • For transcription factor or co-factor ChIP-seq, use at least 5 ng of ChIP-enriched DNA and amplification of the adaptor-ligated DNA with 10 cycles of PCR.
  • For total histone and histone modifications, or input samples, start with 50 ng of ChIP-enriched DNA and amplification of the adaptor-ligated DNA with 6 cycles of PCR.
  • For library construction of ChIP-enriched DNA for all target types, perform cleanup of adaptor-ligated DNA without size selection.
  • After DNA library construction, check the DNA library for presence of adaptor dimers (~140 bp) using an Agilent High Sensitivity DNA Kit (Agilent Technologies, Cat# G2938-90322), or by agarose gel electrophoresis with 50-100 ng DNA on a 2% agarose TAE gel. If adaptor dimers are present, repeat cleanup of PCR amplified material.
  • The quality of the library can also be confirmed using qPCR and primer sets to known positive and negative target loci. Positive primer pairs should still give the same high signal compared to negative primers as seen in the original qPCR analysis of ChIP-enriched DNA.
  • After final cleanup and quality checks, prepare final purified library samples at 2-10 nM for high throughput sequencing.

Appendix A: Expected Chromatin Yield

When harvesting cross-linked chromatin from tissue samples, the yield of chromatin can vary significantly between tissue types. The table to the right provides a range for the expected yield of chromatin from 100 mg of tissue compared to 2 x 107 HCT 116 cells, and the expected DNA concentration, as determined in Section IV of the protocol. For optimal ChIP results, we recommend using 5 to 10 µg of sonicated, cross-linked chromatin per immunoprecipitation.

Tissue/Cell Total Chromatin Yield Expected DNA Concentration
Liver 50 µg per 100 mg tissue 150 µg/ml
Brain 25 µg per 100 mg tissue 50 µg/ml
Heart 105 µg per 100 mg tissue 20 µg/ml
HCT 116 100-150 µg per 2 x 107 cells 100-150 µg/ml

Appendix B: Optimization of Formaldehyde Fixation

Transcription factors and cofactors bind to chromatin DNA more loosely than histone proteins. As a result, they tend to dissociate from chromatin during sonication. Additional fixation time can result in increased capture of transcription factors and cofactors in the ChIP assay, especially with tissue samples. As shown in Figure 7, increasing the fixation time from 10 min to 30 min may reduce chromatin fragmentation size (left panel), but it significantly enhances enrichment of both cofactors RING1B and SUZ12 in heart tissue, as indicated by ChIP-qPCR (middle and right panels).

Typically, 10 min fixation is sufficient for histone modification ChIP with both cell and tissue samples, whereas transcription factors and cofactors may require additional fixation up to 30 min, especially with tissue samples.

FIGURE 7. Mouse heart (H), brain (B), and liver (L) were cross-linked for 10 min or 30 min, as indicated (left panel). The chromatin was prepared and sonicated, DNA was purified and 20 µl was separated by electrophoresis on a 1% agarose gel. In the ChIP-qPCR assay (middle and right panels), chromatin immunoprecipitations were performed with either 10 µl of RING1B (D22F2) XP<sup>®</sup> Rabbit mAb #5694 or 5 µl of SUZ12 (D39F6) XP<sup>®</sup> Rabbit mAb #3737. The enriched DNA was quantified by real-time PCR using SimpleChIP<sup>®</sup> Mouse HoxD10 Exon 1 Primers #7429, SimpleChIP<sup>®</sup> Mouse HoxA1 Promoter Primers #7341, and SimpleChIP<sup>®</sup> Mouse GAPDH Intron 2 Primers #8986. The amount of immunoprecipitated DNA in each sample is represented as normalized signal to the negative GAPDH locus (equivalent to one).

FIGURE 7. Mouse heart (H), brain (B), and liver (L) were cross-linked for 10 min or 30 min, as indicated (left panel). The chromatin was prepared and sonicated, DNA was purified and 20 µl was separated by electrophoresis on a 1% agarose gel. In the ChIP-qPCR assay (middle and right panels), chromatin immunoprecipitations were performed with either 10 µl of RING1B (D22F2) XP® Rabbit mAb #5694 or 5 µl of SUZ12 (D39F6) XP® Rabbit mAb #3737. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse HoxD10 Exon 1 Primers #7429, SimpleChIP® Mouse HoxA1 Promoter Primers #7341, and SimpleChIP® Mouse GAPDH Intron 2 Primers #8986. The amount of immunoprecipitated DNA in each sample is represented as normalized signal to the negative GAPDH locus (equivalent to one).

Appendix C: Optimization of Chromatin Fragmentation

Optimal conditions for the fragmentation of cross-linked chromatin DNA is highly dependent on the number of cells, volume of sample, length of sonication, and sonicator power setting used. For each sonication sample, we recommend using 100-150 mg of tissue or 1 x 107 to 2 x 107 cells per 1 ml ChIP Sonication Nuclear Lysis Buffer. Below is a protocol for determination of the optimal sonication conditions for a specific tissue or cell type.

  1. Prepare cross-linked nuclei from 100 to 150 mg of tissue or 1 x 107 to 2 x 107 cells, as described in Sections I, II, and III. Stop after Step 4 of Section III and proceed as described below.
  2. Fragment chromatin by sonication. Optimal sonication conditions can be determined for a given sonicator by varying the number of rounds or duration of sonication at a given power setting (see Step 5 in Section III for optimal power setting using a Branson Digital Sonifier 250 probe sonicator). To determine optimal sonication conditions, set up a sonication time-course experiment and remove 50 µl samples of chromatin after a given round or duration of sonication. For example, take chromatin samples after each 1 to 2 min of sonication.
  3. Clarify chromatin samples by centrifugation at 21,000 x g in a microcentrifuge for 10 min at 4°C.
  4. Transfer supernatants to new microfuge tubes and add 100 µl nuclease-free water, 6 µl 5 M NaCl #7010, and 2 µl RNAse A #7013. Vortex to mix and incubate samples at 37°C for 30 min.
  5. To each RNase A-digested sample, add 2 µl Proteinase K #10012. Vortex to mix and incubate samples at 65°C for 2 hr.
  6. Remove 20 µl of each sample and determine DNA fragment size
  7. Choose the sonication conditions that generate optimal DNA fragment size and use for chromatin preparation in Step 5 of Section III. If optimal sonication conditions are not achieved, increase or decrease the power setting of the sonicator and repeat the sonication time course.

NOTE: Optimal sonication conditions can vary with different sample types and fixation times. Use the minimal number of sonication cycles required to generate the desired length of chromatin fragments. Over sonication, as indicated by more than 80% of total DNA fragments less than 500 bp, can result in excessive damage to the chromatin and result in lower immunoprecipitation efficiency (see Figure 8, right panel).

  • For sonication of cells fixed for 10 min, optimal sonication conditions will generate a DNA smear with approximately 90% of total DNA fragments less than 1 kb (see Figure 8, left panel). Increasing the fixation time to 30 min will limit fragmentation, generating a DNA smear with approximately 60% of total DNA fragments less than 1 kb.
  • For sonication of tissues fixed for 10 min, optimal sonication conditions will generate a DNA smear with approximately 60% of total DNA fragments less than 1 kb. Increasing the fixation time to 30 min will limit fragmentation, generating a DNA smear with approximately 30% of total DNA fragments less than 1 kb (see Figure 7, left panel).
FIGURE 8. Chromatin immunoprecipitations were performed with 2 x 10<sup>7</sup> HCT 116 cells that were cross-linked for 10 min and sonicated for the time indicated (left panel). DNA was purified as described in Section IV of the SimpleChIP® Plus Sonication Chromatin IP Kit #56383, and 20 µl of purified DNA was separated by electrophoresis on a 1% agarose gel. As shown in the left panel, increasing cycles of sonication reduces the size of chromatin fragments. Chromatin immunoprecipitations were performed with either 5 µl of Non-phospho (Active) Β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb #8814, 10 µl of TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP<sup>®</sup> Plus Sonication Chromatin IP Kit #56383. The enriched DNA was quantified by real-time PCR using SimpleChIP<sup>®</sup> Human CaMK2D Intron 3 Primers #5111 and SimpleChIP<sup>®</sup> Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to one; right panel). As shown, 4 min of chromatin sonication is optimal when using a Branson Digital Sonifier D250 probe sonicator with a 1/8 inch Micro Tip. Over-sonication significantly impairs enrichment of both cofactor beta-catenin and transcription factor TCF4/TCF7L2 containing chromatin.

FIGURE 8. Chromatin immunoprecipitations were performed with 2 x 107 HCT 116 cells that were cross-linked for 10 min and sonicated for the time indicated (left panel). DNA was purified as described in Section IV of the SimpleChIP® Plus Sonication Chromatin IP Kit #56383, and 20 µl of purified DNA was separated by electrophoresis on a 1% agarose gel. As shown in the left panel, increasing cycles of sonication reduces the size of chromatin fragments. Chromatin immunoprecipitations were performed with either 5 µl of Non-phospho (Active) Β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb #8814, 10 µl of TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP® Plus Sonication Chromatin IP Kit #56383. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CaMK2D Intron 3 Primers #5111 and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to one; right panel). As shown, 4 min of chromatin sonication is optimal when using a Branson Digital Sonifier D250 probe sonicator with a 1/8 inch Micro Tip. Over-sonication significantly impairs enrichment of both cofactor beta-catenin and transcription factor TCF4/TCF7L2 containing chromatin.

Appendix D: Troubleshooting Guide

Shorten the crosslinking time within 10-30 minute range. Reduce the amount of cell/tissues per sonication. Conduct sonication time course.
Problem Possible Causes Recommendation
1. Concentration of the fragmented chromatin is too low.

Cell/nuclear lysis is incomplete.

Not enough cells were used for the chromatin preparation.

If DNA concentration of the chromatin preparation is close to 50 µg/ml, add additional chromatin to each IP to give at least 5 µg/IP and continue with protocol.

Count a separate plate of cells before cross-linking to determine an accurate cell number.

2. Chromatin is under-fragmented and fragments are too large (>50% above 1.5kb).

Cells may have been over cross-linked.

Too many cells/tissues were processed.

Shorten the crosslinking time within 10-30 minute range. Reduce the amount of cell/tissues per sonication. Conduct sonication time course.

3. Chromatin is over-fragmented (>90% under 300 bp).

Sonication condition is too harsh.

Conduct a sonication time course to find a minimum output/duration to achieve appropriate sonication.

4. No product or very little product in the input PCR reactions.

Not enough DNA added to the PCR reaction or conditions are not optimal.

PCR amplified region may span nucleosome-free region.

Not enough chromatin added to the IP or chromatin is over-sonicated

Add more DNA to the PCR reaction or increase the number of amplification cycles.

Optimize the PCR conditions for experimental primer set using purified DNA from cross-linked and sonicated chromatin. For optimal ChIP results add 5-10 µg chromatin per IP. See recommendations for problems 1 and 3 above.

5. No product in the positive control Histone H3-IP RPL30 PCR reaction.

Not enough chromatin or antibody added to the IP reaction or IP incubation time is too short.

Incomplete elution of chromatin from Protein G beads.

Be sure to add 5-10 µg of chromatin and 10 µl of antibody to each IP reaction and incubate with antibody over-night and an additional 2 hr after adding Protein G beads.

Elution of chromatin from Protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.

6. Quantity of product in the negative control Rabbit IgG-IP and positive control Histone H3-IP PCR reactions is equivalent.

Too much or not enough chromatin added to the IP reaction. Alternatively, too much antibody added to the IP reaction.

Too much DNA added to the PCR reaction or too many cycles of amplification.

Add no more than 15 µg of chromatin and 10 µl of histone H3 antibody to each IP reaction. Reduce the amount of normal rabbit IgG to 1 µl per IP.

Add less DNA to the PCR reaction or decrease the number of PCR cycles. It is very important that the PCR products are analyzed within the linear amplification phase of PCR. Otherwise, the differences in quantities of starting DNA cannot be accurately measured.

7. No product in the Experimental Antibody-IP PCR reaction.

Not enough DNA added to the PCR reaction.

Not enough antibody added to the IP reaction.

Antibody does not work for IP.

Add more DNA to the PCR reaction or increase the number of amplification cycles.

Typically a range of 1 to 5 µg of antibody is added to the IP reaction; however, the exact amount depends greatly on the individual antibody.

Increase the amount of antibody added to the IP. Find an alternate antibody source.

posted March 2017

Protocol Id: 1404

Specificity / Sensitivity

Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb detects endogenous levels of Stat3 only when phosphorylated at tyrosine 705. This antibody does not cross-react with phospho-EGFR or the corresponding phospho-tyrosines of other Stat proteins.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology:

Hamster, Bovine, Pig, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr705 of mouse Stat3.

Background

The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors (1) and is required for murine fetal development (2). Research studies have shown that Stat3 is constitutively activated in a number of human tumors (3,4) and possesses oncogenic potential (5) and anti-apoptotic activities (3). Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (6,7). Transcriptional activation seems to be regulated by phosphorylation at Ser727 through the MAPK or mTOR pathways (8,9). Stat3 isoform expression appears to reflect biological function as the relative expression levels of Stat3α (86 kDa) and Stat3β (79 kDa) depend on cell type, ligand exposure, or cell maturation stage (10). It is notable that Stat3β lacks the serine phosphorylation site within the carboxy-terminal transcriptional activation domain (8).

  1. Heim, M.H. (1999) J Recept Signal Transduct Res 19, 75-120.
  2. Takeda, K. et al. (1997) Proc Natl Acad Sci U S A 94, 3801-4.
  3. Catlett-Falcone, R. et al. (1999) Immunity 10, 105-15.
  4. Garcia, R. and Jove, R. (1998) J Biomed Sci 5, 79-85.
  5. Bromberg, J.F. et al. (1999) Cell 98, 295-303.
  6. Darnell, J.E. et al. (1994) Science 264, 1415-21.
  7. Ihle, J.N. (1995) Nature 377, 591-4.
  8. Wen, Z. et al. (1995) Cell 82, 241-50.
  9. Yokogami, K. et al. (2000) Curr Biol 10, 47-50.
  10. Biethahn, S. et al. (1999) Exp Hematol 27, 885-94.

Pathways & Proteins

Explore pathways + proteins related to this product.

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