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9167
Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb
Primary Antibodies
Monoclonal Antibody
R
Recombinant

Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167

Citations (989)
Filter:
  1. WB
  2. IP
  3. IHC
  4. IF
  5. F
  6. ChIP
Western blot analysis of extracts from HeLa cells, untreated or treated with IFNa (#36000, 100 ng/mL, 5 min) or IFNg (#80385, 100 ng/mL, 30 min); using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 (upper), Stat1 (D1K9Y) Rabbit mAb #14994 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from various cell lines, untreated or treated with EGF (100 ng/mL, 30 min), IFNa (100 ng/mL, 5 min), or PDGF (100 ng/mL, 5 min); using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 (upper), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from HeLa cells untreated or treated with interferon-α (IFN-α), using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (upper) or Stat1 Antibody (#9172) (lower).
Simple Western™ analysis of lysates (0.1 mg/mL) from serum-starved HeLa cells treated with IFN-alpha (100 ng/mL, 5 min) using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation of Phospho-Stat1 (Tyr701) from HeLa cell extracts, treated with treated with IFNa (#36000, 100 ng/mL, 5 min). Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb, #9167.
Immunohistochemical analysis of paraffin-embedded Non-Hodgkin lymphoma control (left) or λ phosphatase treated (right), using Phospho-Stat1 (tyr701) (58D6) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded Non-Hodgkin lymphoma using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 in the presence of control peptide (left) or Phospho-Stat1 (Tyr701) Blocking Peptide (right).
Immunohistochemical analysis of paraffin-embedded stomach (chronic gastritis), using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb.
Immunohistochemical analysis using Phospho-Stat1 (Tyr701) (58D6) 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)).
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or IFNα-treated #9906 (right), using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with IFN-α (100ng/ml, 5 mins; green) using Phospho-Stat1 (Tyr701) (58D6) 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.
Chromatin immunoprecipitations were performed with cross-linked chromatin from HT-1080 cells treated with IFN-γ (50 ng/ml) for 30 minutes and Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across IRF-1, a known target gene of Phospho-Stat1 (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from HT-1080 cells treated with IFN-γ (50 ng/ml) for 30 minutes and Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 5 (upper), including IRF-1 (lower), a known target gene of Phospho-Stat1 (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from HT-1080 cells treated with IFN-γ (50 ng/ml) for 30 minutes and either Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human IRF-1 promoter primers, SimpleChIP® Human TAP1 Promoter Primers #5148, 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.

Supporting Data

REACTIVITY H M
SENSITIVITY Endogenous
MW (kDa) 84, 91
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • 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
  • GP-Guinea Pig
  • Rab-Rabbit
  • 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 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:400 - 1:1600
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800
Flow Cytometry (Fixed/Permeabilized) 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.

For a carrier-free (BSA and azide free) version of this product see product #88845.

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. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  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 (#59329, 10 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 for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.

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 Magnetic Beads: (#73778).
  5. Magnetic Separation Rack: (#7017) or (#14654).
  6. 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.
  7. 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)

A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.

  1. Briefly vortex the stock tube to resuspend the magnetic beads.

    IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:

  2. Transfer 20 μl of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 seconds.

    Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.

  3. Add 200 μl cell lysate to 20 μl of pre-washed magnetic beads.

    IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.

  4. Incubate with rotation for 20 minutes at room temperature.
  5. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.
  6. Proceed to immunoprecipitation section.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate. Incubate with rotation overnight at 4°C. to form the immunocomplex.
  2. Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2).
  3. Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet.
  4. Incubate with rotation for 20 min at room temperature.
  5. Pellet beads using magnetic separation rack. Wash pellets five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes.
  6. Proceed to analyze 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 20-40 µl 3X SDS sample buffer, briefly vortex to mix, and briefly microcentrifuge to pellet the sample.
  2. Heat the sample to 95-100°C for 5 min.
  3. Pellet beads using magnetic separation rack. Transfer the supernatant to a new tube. The supernatant is the sample.
  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) (D4W3E) mAb (#45262) 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 gel.

posted December 2008

revised April 2021

Protocol Id: 410

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 SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824
SignalStain® Deep Black Peroxidase Substrate Kit #72986  
SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644  

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

Chromatin IP

Specific for product: SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005.

Required Reagents

Reagents Included:

  1. Glycine Solution (10X) #7005
  2. Buffer A (4X) #7006
  3. Buffer B (4X) #7007
  4. ChIP Buffer (10X) #7008
  5. ChIP Elution Buffer (2X) #7009
  6. 5 M NaCl #7010
  7. 0.5 M EDTA #7011
  8. ChIP-Grade Protein G Magnetic Beads #9006
  9. DNA Binding Buffer #10007
  10. DNA Wash Buffer (add 4x volume ethanol before use) #10008
  11. DNA Elution Buffer #10009
  12. DNA Purification Columns and Collection Tubes #10010
  13. Protease Inhibitor Cocktail (200X) #7012
  14. RNAse A (10 mg/ml) #7013
  15. Micrococcal Nuclease #10011
  16. Proteinase K (20 mg/ml) #10012
  17. SimpleChIP® Human RPL30 Exon 3 Primers 1 #7014
  18. SimpleChIP® Mouse RPL30 Intron 2 Primers 1 #7015
  19. Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620
  20. Normal Rabbit IgG #2729
  21. DTT (Dithiothreitol) #7016

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. Ethanol (96-100%)
  5. Formaldehyde (37% Stock)
  6. SimpleChIP® Universal qPCR Master Mix #88989
! 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 such as fat and necrotic material from the sample. Tissue can then be processed and cross-linked immediately, or frozen on dry ice and stored at -80°C for processing later. For optimal chromatin yield and ChIP results, use 25 mg of tissue for each immunoprecipitation to be performed. The chromatin yield does vary between tissue types and some tissues may require more than 25 mg for each immunoprecipitation. Please see Appendix A for more information regarding the expected chromatin yield for different types of tissue. One additional chromatin sample should be processed for Analysis of Chromatin Digestion and Concentration (Section IV). If desired, five additional chromatin samples should be processed for Optimization of Chromatin Digestion (Appendix B).

Before starting:

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

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed.
  • Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 µl 200X PIC per 25 mg of tissue to be processed and place on ice.
  • Prepare 45 µl of 37% formaldehyde per 25 mg of tissue to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.

A. Cross-linking

  1. Weigh the fresh or frozen tissue sample. Use 25 mg of tissue for each IP to be performed (at least 75 mg of tissue is required for one experiment in order to include positive and negative controls).
  2. Place tissue sample in a 60 mm or 100 mm dish and finely mince 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.
  4. Add 1 ml of PBS + PIC per 25 mg tissue to the conical tube.
  5. To crosslink proteins to DNA, add 45 µl of 37% formaldehyde per 1 ml of PBS + PIC and rock at room temp for 20 min. Final formaldehyde concentration is 1.5%.
  6. Stop cross-linking by adding 100 µl of 10X Glycine per 1 ml of PBS + PIC and mix for 5 min at room temperature.
  7. Centrifuge tissue at 500 x g in a benchtop centrifuge for 5 min at 4°C.
  8. Remove supernatant and wash one time with 1 ml PBS + PIC per 25 mg tissue.
  9. Repeat centrifugation at 500 x g in a benchtop centrifuge for 5 min at 4°C.
  10. Remove supernatant and resuspend tissue in 1 ml PBS + PIC per 25 mg tissue and store on ice. Disaggregate tissue into single-cell suspension using a Medimachine (Part B) or Dounce homogenizer (Part C). (SAFE STOP) Alternatively, samples may be stored at -80°C before disaggregation for up to 3 months.

B. Tissue Disaggregation Using Medimachine from BD Biosciences (part #340587)

  1. Cut off the end of a 1000 µL pipette tip to enlarge the opening for transfer of tissue chunks.
  2. Transfer 1 ml of tissue resuspended in PBS + PIC into the top chamber of a 50 mm medicone (part #340592).
  3. Grind tissue for 2 min according to manufacturer's instructions.
  4. Collect cell suspension from the bottom chamber of the medicone using a 1 ml syringe and 18 gauge blunt needle. Transfer cell suspension to a 15 ml conical tube and place on ice.
  5. Repeat steps 2 to 4 until all the tissue is processed into a homogenous suspension.
  6. If more grinding is necessary, add more PBS + PIC to tissue. Repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension.
  7. Check for single-cell suspension by microscope (optional).
  8. Centrifuge cells at 2,000 x g in a bench top centrifuge for 5 min at 4°C.
  9. Remove supernatant from cells and continue with Nuclei Preparation and Chromatin Digestion (Section III).

C. Tissue Disaggregation Using a Dounce Homogenizer

  1. Transfer tissue resuspended in PBS + PIC to a Dounce homogenizer.
  2. Disaggregate tissue pieces with 20-25 strokes. Check for single-cell suspension by microscope (optional).
  3. Transfer cell suspension to a 15 ml conical tube and centrifuge at 2,000 x g in a benchtop centrifuge for 5 min at 4°C.
  4. Remove supernatant from cells and continue with Nuclei Preparation and Chromatin Digestion (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 (at least 12 X 106 cells are required in order to include positive and negative controls). For HeLa cells, one IP is equivalent to half of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. One additional sample should be processed for Analysis of Chromatin Digestion and Concentration (Section IV). Since every cell type is different, we recommend including one extra dish of cells in experiment to be used for determination of cell number using a hemocytometer or cell counter.

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.

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and 10X Glycine Solution #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 (or 20 ml suspension cells) to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.
  1. To crosslink proteins to DNA, add 540 µl of 37% formaldehyde to each 15 cm culture dish containing 20 ml medium. For suspension cells, add 540 µl of 37% formaldehyde to cells suspended in 20 ml medium (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, transfer cells to a 50 ml conical tube, centrifuge at 500 x g in a benchtop centrifuge 5 min at 4°C and wash pellet two times with 20 ml ice-cold PBS. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).
  4. For adherent cells, remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from culture dish each time.
  5. 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.
  6. Centrifuge cells at 2,000 x g in a benchtop centrifuge for 5 min at 4°C. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III). (SAFE STOP) Alternatively samples may be stored at -80°C for up to 3 months.

III. Nuclei Preparation and Chromatin Digestion

Before starting

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

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure it is completely thawed prior to use.
  • Prepare 1 M DTT (192.8 mg DTT #7016 + 1.12ml dH2O). Make sure DTT crystals are completely in solution.

    (!!) IMPORTANT: Once in solution, store 1M DTT at -20°C.

  • Remove and warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.
  • Prepare 1 ml 1X Buffer A (250 µl 4X Buffer A #7006 + 750 µl water) + 0.5 µl 1M DTT + 5 µl 200X PIC per IP prep and place on ice.
  • Prepare 1.1 ml 1X Buffer B (275 µl 4X Buffer B #7007 + 825 µl water) + 0.55 µl 1M DTT per IP prep and place on ice.
  • Prepare 100 µl 1X ChIP Buffer (10 µl 10X ChIP Buffer #7008 + 90 µl water) + 0.5 µl 200X PIC per IP prep and place on ice.
  1. Resuspend cells in 1 ml ice-cold 1X Buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting tube every 3 min.
  2. Pellet nuclei by centrifugation at 2,000 x g in a benchtop centrifuge for 5 min at 4°C. Remove supernatant and resuspend pellet in 1 ml ice-cold 1X Buffer B + DTT per IP prep. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 µl 1X Buffer B +DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to 1 ml total per tube.
  3. Add 0.5 µl of Micrococcal Nuclease #10011 per IP prep, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3 to 5 min. The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined empirically for individual tissues and cell lines (see Appendix B). HeLa nuclei digested with 0.5 µl Micrococcal Nuclease per 4 x 106 cells and mouse liver tissue digested with 0.5 µl Micrococcal Nuclease per 25 mg of tissue gave the appropriate length DNA fragments.
  4. Stop digest by adding 10 µl of 0.5 M EDTA #7011 per IP prep and placing tube on ice for 1-2 min.
  5. Pellet nuclei by centrifugation at 16,000 x g in a microcentrifuge for 1 min at 4°C and remove supernatant.
  6. Resuspend nuclear pellet in 100 µl of 1X ChIP Buffer + PIC per IP prep and incubate on ice for 10 min.
  7. Sonicate up to 500 µl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  8. Clarify lysates by centrifugation at 9,400 x g in a microcentrifuge for 10 min at 4°C.
  9. 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). This 50 µl sample may be stored at -20°C overnight.

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

  1. To the 50 µl chromatin sample (from Step 9 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. Vortex to mix and incubate samples at 65°C for 2 h.
  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 with a 100 bp DNA marker. DNA should be digested to a length of approximately 150-900 bp (1 to 5 nucleosomes).
  5. To determine DNA concentration, transfer 2 µl of purified DNA to 98 µl nuclease-free water to give a 50-fold dilution and read the OD260. The concentration of DNA in µg/ml is OD260 x 2,500. DNA concentration should ideally be between 50 and 200 µg/ml.

NOTE: For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. Over-digestion of chromatin may diminish signal in the PCR quantification. Under-digestion of chromatin may lead to increased background signal and lower resolution. Adding too little chromatin to the IP may result in diminished signal in the PCR quantification. A protocol for optimization of chromatin digestion can be found in Appendix B.

V. Chromatin Immunoprecipitation

For optimal ChIP results, use approximately 5 to 10 µg of digested, 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 4 x 106 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 does not need to be diluted as described below. Antibodies can be added directly to the undiluted chromatin preparation for immunoprecipitation of chromatin complexes.

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 digested chromatin preparation (from Step 9 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. Store at room temperature until use.
  • 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. Store at room temperature until use.
  1. In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin into the desired number of immunoprecipitations: 400 µl of 1X ChIP Buffer (40 µl of 10X ChIP Buffer + 360 µl water) + 2 µl 200X PIC per immunoprecipitation. 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. Place mix on ice.
  2. To the prepared 1X ChIP Buffer, add the equivalent of 100 µl (5 to 10 µg of chromatin) of the digested, cross-linked chromatin preparation (from Step 9 in Section III) per immunoprecipitation. For example, for 10 immunoprecipitations, prepare a tube containing 4 ml 1X ChIP Buffer (400 µl 10X ChIP Buffer + 3.6 ml water) + 20 µl 200X PIC + 1 ml digested chromatin preparation.
  3. 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).
  4. For each immunoprecipitation, transfer 500 µl of the diluted chromatin to a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody required per IP varies and should be determined by the user. 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. If using antibodies from Cell Signaling Technology, please see recommended dilution listed on the datasheet or product webpage and calculate the amount (µg) of IgG antibody for negative control based on the Cell Signaling Antibody concentration for a fair comparison. Incubate IP samples 4 h to overnight at 4°C with rotation.

    NOTE: Most antibodies from Cell Signaling Technology work optimally between 1 and 2 ug 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 h at 4°C with rotation.
  6. Pellet protein G magnetic beads in each immunoprecipitation by placing the tubes in a magnetic separation rack #7017. 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

(!) 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 sample.
  1. Add 150 µl of the 1X ChIP Elution Buffer to the 2% input sample tube and set aside at room temperature until Step 6.
  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. Alternatively, elutions can be performed at room temperature with rotation, but may not be as complete.
  4. Pellet protein G magnetic beads by placing the tubes in a magnetic separation rack and wait 1 to 2 min for solution to clear.
  5. Carefully transfer eluted chromatin supernatant to a new tube.
  6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 µl 5M NaCl and 2 µl Proteinase K #10012, and incubate 2 h at 65°C. This incubation can be extended overnight.
  7. 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.
  • Remove one DNA Purification collection tube #10010 for each DNA sample from Section V.
  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 sample from Step 1 to a DNA spin column in collection tube.
  3. Centrifuge at 18,500 x g 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 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 18,500 x g 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 18,500 x g 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 18,000 x g 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.

VIII. Quantification of DNA by PCR

Recommendations

  • Use Filter-tip pipette tips to minimize risk of contamination.
  • The control primers included in the kit are specific for the human or mouse RPL30 gene (#7014 + #7015) and can be used for either standard PCR or quantitative real-time PCR. If the user is performing ChIPs 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 0.2 ml PCR tubes for the number of samples to be analyzed. 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
  1. Start the following PCR reaction program:
a. Initial Denaturation 95°C 5 min
b. Denature 95°C 30 sec
c. Anneal 62°C 30 sec
d. Extension 72°C 30 sec
e. Repeat Steps b-d for a total of 34 cycles.
f. Final Extension 72°C 5 min
  1. 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. Add enough reagents for two extra reactions 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
  1. 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.
  1. 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 = 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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 and its associated index primers Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #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 in the DNA library, 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 25 mg of tissue compared to 4 x 106 HeLa cells, and the expected DNA concentration, as determined in Section IV of the protocol. For each tissue type, disaggregation using a Medimachine (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, chromatin processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension. For optimal ChIP results, we recommend using 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per each immunoprecipitation.

Tissue/Cell Total Chromatin Yield Expected DNA Concentration
Spleen 20-30 µg per 25 mg tissue 200-300 µg/ml
Liver 10-15 µg per 25 mg tissue 100-150 µg/ml
Kidney 8-10 µg per 25 mg tissue 80-100 µg/ml
Brain 2-5 µg per 25 mg tissue 20-50 µg/ml
Heart 2-5 µg per 25 mg tissue 20-50 µg/ml
HeLa 10-15 µg per 4 x 106 cells 100-150 µg/ml

APPENDIX B: Optimization of Chromatin Digestion

Optimal conditions for the digestion of cross-linked chromatin DNA to 150-900 base pairs in length is highly dependent on the ratio of Micrococcal Nuclease to the amount of tissue or number of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific tissue or cell type.

  1. Prepare cross-linked nuclei from 125 mg of tissue or 2 X 107 cells (equivalent of 5 IP preps), as described in Sections I, II, and III. Stop after Step 2 of Section III and proceed as described below.
  2. Transfer 100 µl of the nuclei preparation into 5 individual 1.5 ml microcentrifuge tubes and place on ice.
  3. Add 3 µl Micrococcal Nuclease stock to 27 µl of 1X Buffer B + DTT (1:10 dilution of enzyme).
  4. To each of the 5 tubes in Step 2, add 0 µl, 2.5 µl, 5 µl, 7.5 µl, or 10 µl of the diluted Micrococcal Nuclease, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing.
  5. Stop each digest by adding 10 µl of 0.5 M EDTA and placing tubes on ice.
  6. Pellet nuclei by centrifugation at 16,000 x g in a microcentrifuge for 1 min at 4°C and remove supernatant.
  7. Resuspend nuclear pellet in 200 µl of 1X ChIP Buffer + PIC. Incubate on ice for 10 min.
  8. Sonicate lysate with several pulses to break nuclear membrane. Incubate samples 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator set at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  9. Clarify lysates by centrifugation at 9,400 x g in a microcentrifuge for 10 min at 4°C.
  10. Transfer 50 µl of each of the sonicated lysates to new microfuge tubes.
  11. To each 50 µl sample, add 100 µl nuclease-free water, 6 µl 5 M NaCl and 2 µl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
  12. To each RNAse A-digested sample, add 2 µl Proteinase K. Vortex to mix and incubate sample at 65°C for 2 h.
  13. Remove 20 µl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker.
  14. Observe which of the digestion conditions produces DNA in the desired range of 150-900 base pairs (1 to 5 nucleosomes). The volume of diluted Micrococcal Nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to 10 times the volume of Micrococcal Nuclease stock that should be added to one immunoprecipitation preparation (25 mg of disaggregated tissue cells or 4 X 106 tissue culture cells) to produce the desired size of DNA fragments. For example, if 5 µl of diluted Micrococcal Nuclease produces DNA fragments of 150-900 base pairs in this protocol, then 0.5 µl of stock Micrococcal Nuclease should be added to one IP prep during the digestion of chromatin in Section III.
  15. If results indicate that DNA is not in the desired size range, then repeat optimization protocol, adjusting the amount of Micrococcal Nuclease in each digest accordingly. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.

APPENDIX C: Troubleshooting Guide

Problem Possible Causes Recommendation
1. Concentration of the digested chromatin is too low. Not enough cells added to the chromatin digestion or nuclei were not completely lysed after digestion.

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 and/or visualize nuclei under microscope before and after sonication to confirm complete lysis of nuclei.

2. Chromatin is under-digested and fragments are too large (greater than 900 bp).

Cells may have been over cross-linked. Cross-linking for longer than 10 min may inhibit digestion of chromatin.

Too many cells or not enough Micrococcal Nuclease was added to the chromatin digestion.

Perform a time course at a fixed formaldehyde concentration. Shorten the time of cross-linking to 10 min or less.

Count a separate plate of cells before cross-linking to determine accurate cell number and see Appendix B for optimization of chromatin digestion.

3. Chromatin is over-digested and fragments are too small (exclusively 150 bp mono-nucleosome length). Complete digestion of chromatin to mono-nucleosome length DNA may diminish signal during PCR quantification, especially for amplicons greater than 150 bp in length. Not enough cells or too much Micrococcal Nuclease added to the chromatin digestion. Count a separate plate of cells before cross-linking to determine accurate cell number and see Appendix B for optimization of chromatin digestion.
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-digested.

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 digested chromatin. Design a different primer set and decrease length of amplicon to less than 150 bp (see primer design recommendations in Section VIII).

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 h 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 can not 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 are 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 December 2011

revised April 2022

Protocol Id: 82

Chromatin IP

Specific for product: SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005.

Required Reagents

Reagents Included:

  1. Glycine Solution (10X) #7005
  2. Buffer A (4X) #7006
  3. Buffer B (4X) #7007
  4. ChIP Buffer (10X) #7008
  5. ChIP Elution Buffer (2X) #7009
  6. 5 M NaCl #7010
  7. 0.5 M EDTA #7011
  8. ChIP-Grade Protein G Magnetic Beads #9006
  9. DNA Binding Buffer #10007
  10. DNA Wash Buffer (add 4x volume ethanol before use) #10008
  11. DNA Elution Buffer #10009
  12. DNA Purification Columns #10010
  13. Protease Inhibitor Cocktail (200X) #7012
  14. RNAse A (10 mg/ml) #7013
  15. Micrococcal Nuclease (2000 gel units/µl) #10011
  16. Proteinase K (20 mg/ml) #10012
  17. SimpleChIP® Human RPL30 Exon 3 Primers 1 #7014
  18. SimpleChIP® Mouse RPL30 Intron 2 Primers 1 #7015
  19. Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620
  20. Normal Rabbit IgG #2729
  21. DTT (Dithiothreitol) #7016

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. Ethanol (96-100%)
  5. Formaldehyde (37% Stock)
  6. Taq DNA Polymerase
  7. dNTP Mix

I. Tissue Cross-linking and Sample Preparation

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

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed.
  • Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 µl 200X PIC per 25 mg of tissue to be processed and place on ice.
  • Prepare 45 µl of 37% formaldehyde per 25 mg of tissue to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.

A. Cross-linking

  1. Weigh the fresh or frozen tissue sample. Use 25 mg of tissue for each IP to be performed.
  2. Place tissue sample in a 60 mm or 100 mm dish and finely mince 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.
  4. Add 1 ml of PBS + PIC per 25 mg tissue to the conical tube.
  5. To crosslink proteins to DNA, add 45 µl of 37% formaldehyde per 1 ml of PBS + PIC and rock at room temp for 20 min. Final formaldehyde concentration is 1.5%.
  6. Stop cross-linking by adding 100 µl of 10X Glycine per 1 ml of PBS + PIC and mix for 5 min at room temperature.
  7. Centrifuge tissue at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C.
  8. Remove supernatant and wash one time with 1 ml PBS + PIC per 25 mg tissue.
  9. Repeat centrifugation at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C.
  10. Remove supernatant and resuspend tissue in 1 ml PBS + PIC per 25 mg tissue and store on ice. Disaggregate tissue into single-cell suspension using a Medimachine (Part B) or Dounce homogenizer (Part C).

B. Tissue Disaggregation Using Medimachine from BD Biosciences (part #340587)

  1. Cut off the end of a 1000 µL pipette tip to enlarge the opening for transfer of tissue chunks.
  2. Transfer 1 ml of tissue resuspended in PBS + PIC into the top chamber of a 50 mm medicone (part #340592).
  3. Grind tissue for 2 min according to manufacturer's instructions.
  4. Collect cell suspension from the bottom chamber of the medicone using a 1 ml syringe and 18 gauge blunt needle. Transfer cell suspension to a 15 ml conical tube and place on ice.
  5. Repeat steps 2 to 4 until all the tissue is processed into a homogenous suspension.
  6. If more grinding is necessary, add more PBS + PIC to tissue. Repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension.
  7. Check for single-cell suspension by microscope (optional).
  8. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  9. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

C. Tissue Disaggregation Using a Dounce Homogenizer

  1. Transfer tissue resuspended in PBS + PIC to a Dounce homogenizer.
  2. Disaggregate tissue pieces with 20-25 strokes. Check for single-cell suspension by microscope (optional).
  3. Transfer cell suspension to a 15 ml conical tube and centrifuge at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C.
  4. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (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 HeLa cells, this is equivalent to half of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. One additional sample should be processed for Analysis of Chromatin Digestion and Concentration (Section IV). Include one extra dish of cells in experiment to be used for determination of cell number using a hemocytometer.

Before starting

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and 10X Glycine Solution #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. Use fresh formaldehyde that is not past the manufacturer's expiration date.
  1. To crosslink proteins to DNA, add 540 µl of 37% 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, transfer cells to a 50 ml conical tube, centrifuge at 1,500 rpm in a benchtop centrifuge 5 min at 4°C and wash pellet two times with 20 ml ice-cold PBS. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).
  4. For adherent cells, remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from culture dish each time.
  5. 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.
  6. Centrifuge cells at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

III. Nuclei Preparation and Chromatin Digestion

One immunoprecipitation preparation (IP prep) is defined as 25 mg of disaggregated tissue or 4 x 106 tissue culture cells.

Before starting

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure it is completely thawed prior to use.
  • Prepare 1 M DTT (192.8 mg DTT #7016 + 1.12ml dH2O). Make sure DTT crystals are completely in solution.

    IMPORTANT: Once in solution, store 1M DTT at -20°C.

  • Remove and warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.
  • Prepare 1 ml 1X Buffer A (250 µl 4X Buffer A #7006 + 750 µl water) + 0.5 µl 1M DTT + 5 µl 200X PIC per IP prep and place on ice.
  • Prepare 1.1 ml 1X Buffer B (275 µl 4X Buffer B #7007 + 825 µl water) + 0.55 µl 1M DTT per IP prep and place on ice.
  • Prepare 100 µl 1X ChIP Buffer (10 µl 10X ChIP Buffer #7008 + 90 µl water) + 0.5 µl 200X PIC per IP prep and place on ice.
  1. Resuspend cells in 1 ml ice-cold 1X Buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting tube every 3 min.
  2. Pellet nuclei by centrifugation at 3,000 rpm in a benchtop centrifuge for 5 min at 4°C. Remove supernatant and resuspend pellet in 1 ml ice-cold 1X Buffer B + DTT per IP prep. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 µl 1X Buffer B +DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to 1 ml total per tube.
  3. Add 0.5 µl of Micrococcal Nuclease #10011 per IP prep, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3 to 5 min. The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined empirically for individual tissues and cell lines (see Appendix B). HeLa nuclei digested with 0.5 µl Micrococcal Nuclease per 4 x 106 cells and mouse liver tissue digested with 0.5 µl Micrococcal Nuclease per 25 mg of tissue gave the appropriate length DNA fragments.
  4. Stop digest by adding 10 µl of 0.5 M EDTA #7011 per IP prep and placing tube on ice.
  5. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
  6. Resuspend nuclear pellet in 100 µl of 1X ChIP Buffer + PIC per IP prep and incubate on ice for 10 min.
  7. Sonicate up to 500 µl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  8. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
  9. Transfer supernatant to a new tube. 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 Digestion and Concentration (Recommended Step)

  1. To the 50 µl chromatin sample (from Step 9 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. Vortex to mix and incubate samples at 65°C for 2 h.
  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 with a 100 bp DNA marker. DNA should be digested to a length of approximately 150-900 bp (1 to 5 nucleosomes).
  5. To determine DNA concentration, transfer 2 µl of purified DNA to 98 µl nuclease-free water to give a 50-fold dilution and read the OD260. The concentration of DNA in µg/ml is OD260 x 2,500. DNA concentration should ideally be between 50 and 200 µg/ml.

NOTE: For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. Over-digestion of chromatin may diminish signal in the PCR quantification. Under-digestion of chromatin may lead to increased background signal and lower resolution. Adding too little chromatin to the IP may result in diminished signal in the PCR quantification. A protocol for optimization of chromatin digestion can be found in Appendix B.

V. Chromatin Immunoprecipitation

For optimal ChIP results, use approximately 5 to 10 µg of digested, 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 4 x 106 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 does not need to be diluted as described below. Antibodies can be added directly to the undiluted chromatin preparation for immunoprecipitation of chromatin complexes.

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 digested chromatin preparation (from Step 9 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. Store at room temperature until use.
  • 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. Store at room temperature until use.
  1. In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin into the desired number of immunoprecipitations: 400 µl of 1X ChIP Buffer (40 µl of 10X ChIP Buffer + 360 µl water) + 2 µl 200X PIC per immunoprecipitation. 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. Place mix on ice.
  2. To the prepared 1X ChIP Buffer, add the equivalent of 100 µl (5 to 10 µg of chromatin) of the digested, cross-linked chromatin preparation (from Step 9 in Section III) per immunoprecipitation. For example, for 10 immunoprecipitations, prepare a tube containing 4 ml 1X ChIP Buffer (400 µl 10X ChIP Buffer + 3.6 ml water) + 20 µl 200X PIC + 1 ml digested chromatin preparation.
  3. 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).
  4. For each immunoprecipitation, transfer 500 µl of the diluted chromatin to a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody required per IP varies and should be determined by the user. 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 h to overnight at 4°C with rotation.

NOTE: Most antibodies from Cell Signaling Technology work optimally between 1 and 2 ug 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.

  1. 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 h at 4°C with rotation.
  2. Pellet protein G magnetic beads in each immunoprecipitation by placing the tubes in a magnetic separation rack #7017. Wait 1 to 2 min for solution to clear and then carefully remove supernatant.
  3. 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.
  4. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
  5. 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 sample.
  1. Add 150 µl of the 1X ChIP Elution Buffer to the 2% input sample tube and set aside at room temperature until Step 6.
  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. Alternatively, elutions can be performed at room temperature with rotation, but may not be as complete.
  4. Pellet protein G magnetic beads by placing the tubes in a magnetic separation rack and wait 1 to 2 min for solution to clear.
  5. Carefully transfer eluted chromatin supernatant to a new tube.
  6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 µl 5M NaCl and 2 µl Proteinase K #10012, and incubate 2 h at 65°C. This incubation can be extended overnight.
  7. 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.
  • Remove one DNA Purification collection tube #10010 for each DNA sample from Section V.
  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 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 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 Filter-tip pipette tips to minimize risk of contamination.
  • The control primers included in the kit are specific for the human or mouse RPL30 gene (#7014 + #7015) and can be used for either standard PCR or quantitative real-time PCR. If the user is performing ChIPs 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 0.2 ml PCR tubes for the number of samples to be analyzed. 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
  1. Start the following PCR reaction program:
a. Initial Denaturation 95°C 5 min
b. Denature 95°C 30 sec
c. Anneal 62°C 30 sec
d. Extension 72°C 30 sec
e. Repeat Steps b-d for a total of 34 cycles.
f. Final Extension 72°C 5 min
  1. 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. Add enough reagents for two extra reactions 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
  1. 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.
  1. 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 = 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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 and its associated index primers Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #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 in the DNA library, 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 25 mg of tissue compared to 4 x 106 HeLa cells, and the expected DNA concentration, as determined in Section IV of the protocol. For each tissue type, disaggregation using a Medimachine (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, chromatin processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension. For optimal ChIP results, we recommend using 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per each immunoprecipitation.

Tissue/Cell Total Chromatin Yield Expected DNA Concentration
Spleen 20-30 µg per 25 mg tissue 200-300 µg/ml
Liver 10-15 µg per 25 mg tissue 100-150 µg/ml
Kidney 8-10 µg per 25 mg tissue 80-100 µg/ml
Brain 2-5 µg per 25 mg tissue 20-50 µg/ml
Heart 2-5 µg per 25 mg tissue 20-50 µg/ml
HeLa 10-15 µg per 4 x 106 cells 100-150 µg/ml

APPENDIX B: Optimization of Chromatin Digestion

Optimal conditions for the digestion of cross-linked chromatin DNA to 150-900 base pairs in length is highly dependent on the ratio of Micrococcal Nuclease to the amount of tissue or number of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific tissue or cell type.

  1. Prepare cross-linked nuclei from 125 mg of tissue or 2 X 107 cells (equivalent of 5 IP preps), as described in Sections I, II, and III. Stop after Step 2 of Section III and proceed as described below.
  2. Transfer 100 µl of the nuclei preparation into 5 individual 1.5 ml microcentrifuge tubes and place on ice.
  3. Add 3 µl Micrococcal Nuclease stock to 27 µl of 1X Buffer B + DTT (1:10 dilution of enzyme).
  4. To each of the 5 tubes in Step 2, add 0 µl, 2.5 µl, 5 µl, 7.5 µl, or 10 µl of the diluted Micrococcal Nuclease, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing.
  5. Stop each digest by adding 10 µl of 0.5 M EDTA and placing tubes on ice.
  6. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
  7. Resuspend nuclear pellet in 200 µl of 1X ChIP Buffer + PIC. Incubate on ice for 10 min.
  8. Sonicate lysate with several pulses to break nuclear membrane. Incubate samples 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator set at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  9. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
  10. Transfer 50 µl of each of the sonicated lysates to new microfuge tubes.
  11. To each 50 µl sample, add 100 µl nuclease-free water, 6 µl 5 M NaCl and 2 µl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
  12. To each RNAse A-digested sample, add 2 µl Proteinase K. Vortex to mix and incubate sample at 65°C for 2 h.
  13. Remove 20 µl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker.
  14. Observe which of the digestion conditions produces DNA in the desired range of 150-900 base pairs (1 to 5 nucleosomes). The volume of diluted Micrococcal Nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to 10 times the volume of Micrococcal Nuclease stock that should be added to one immunoprecipitation preparation (25 mg of disaggregated tissue cells or 4 X 106 tissue culture cells) to produce the desired size of DNA fragments. For example, if 5 µl of diluted Micrococcal Nuclease produces DNA fragments of 150-900 base pairs in this protocol, then 0.5 µl of stock Micrococcal Nuclease should be added to one IHC prep during the digestion of chromatin in Section III.
  15. If results indicate that DNA is not in the desired size range, then repeat optimization protocol, adjusting the amount of Micrococcal Nuclease in each digest accordingly. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.

APPENDIX C: Troubleshooting Guide

Problem Possible Causes Recommendation
1. Concentration of the digested chromatin is too low. Not enough cells added to the chromatin digestion or nuclei were not completely lysed after digestion.

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 and/or visualize nuclei under microscope before and after sonication to confirm complete lysis of nuclei.

2. Chromatin is under-digested and fragments are too large (greater than 900 bp).

Cells may have been over cross-linked. Cross-linking for longer than 10 min may inhibit digestion of chromatin.

Too many cells or not enough Micrococcal Nuclease was added to the chromatin digestion.

Perform a time course at a fixed formaldehyde concentration. Shorten the time of cross-linking to 10 min or less.

Count a separate plate of cells before cross-linking to determine accurate cell number and see Appendix B for optimization of chromatin digestion.

3. Chromatin is over-digested and fragments are too small (exclusively 150 bp mono-nucleosome length). Complete digestion of chromatin to mono-nucleosome length DNA may diminish signal during PCR quantification, especially for amplicons greater than 150 bp in length. Not enough cells or too much Micrococcal Nuclease added to the chromatin digestion. Count a separate plate of cells before cross-linking to determine accurate cell number and see Appendix B for optimization of chromatin digestion.
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-digested.

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 digested chromatin. Design a different primer set and decrease length of amplicon to less than 150 bp (see primer design recommendations in Section VIII).

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 h 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 can not 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 are 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 December 2011

revised April 2022

Protocol Id: 1184

Specificity / Sensitivity

Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb detects endogenous levels of Stat1 only when phosphorylated at tyrosine 701. The antibody detects phosphorylated tyrosine 701 of p91 Stat1 and also the p84 splice variant. It does not cross-react with the corresponding phospho-tyrosines of other Stat proteins.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr701 of human Stat1.

Background

The Stat1 transcription factor is activated in response to a large number of ligands (1) and is essential for responsiveness to IFN-α and IFN-γ (2,3). Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization, nuclear translocation, and DNA binding (4). Stat1 protein exists as a pair of isoforms, Stat1α (91 kDa) and the splice variant Stat1β (84 kDa). In most cells, both isoforms are activated by IFN-α, but only Stat1α is activated by IFN-γ. The inappropriate activation of Stat1 occurs in many tumors (5). In addition to tyrosine phosphorylation, Stat1 is also phosphorylated at Ser727 through a p38 mitogen-activated protein kinase (MAPK)-dependent pathway in response to IFN-α and other cellular stresses (6). Serine phosphorylation may be required for the maximal induction of Stat1-mediated gene activation.

Pathways

Explore pathways related to this product.

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