Product # | Size | Price |
---|---|---|
32578S | 100 µl | $ 276 |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | 130, 95, 85 |
Source/Isotype | Rabbit IgG |
Product Information
For optimal ChIP and ChIP-seq results, use 10 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.
The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
Immunofluorescence (Immunocytochemistry) | 1:1600 |
Flow Cytometry | 1:1600 |
Chromatin IP | 1:50 |
Chromatin IP-seq | 1:50 |
CUT&RUN | 1:50 |
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 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.
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.
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.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
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.
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.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
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.
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.
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, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples
Proceed to one of the following specific set of steps.
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).
posted December 2008
revised April 2018
Protocol Id: 410
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
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.
posted November 2006
revised November 2013
Protocol Id: 24
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.
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.
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.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 404
Specific for product: SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005.
Reagents Included:
Reagents Not Included:
! | 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. |
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).
(!) All buffer volumes should be increased proportionally based on the number of IP preps in the experiment.
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.
(!) All buffer volumes should be increased proportionally based on the number of 15 cm tissue culture dishes (or 20 ml suspension cells) used.
(!) All buffer volumes should be increased proportionally based on the number of IP preps in the experiment.
(!!) IMPORTANT: Once in solution, store 1M DTT at -20°C.
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.
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.
(!) All buffer volumes should be increased proportionally based on the number of immunoprecipitations in the experiment.
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.
(!) All buffer volumes should be increased proportionally based on the number of immunoprecipitations in the experiment.
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) |
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 |
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 |
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 |
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. |
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
The immuno-enriched DNA samples prepared with this kit are directly compatible with ChIP-seq. For downstream NG-sequencing DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina® platforms, we recommend SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 and its associated index primers SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers) #29580 or SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers) #47538.
Recommendations:
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 |
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.
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 June 2018
Protocol Id: 82
Specific for product: SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005.
Reagents Included:
Reagents Not Included:
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).
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.
One immunoprecipitation preparation (IP prep) is defined as 25 mg of disaggregated tissue or 4 x 106 tissue culture cells.
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.
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.
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.
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.
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) |
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 |
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 |
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 |
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. |
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
The immuno-enriched DNA samples prepared with this kit are directly compatible with ChIP-seq. For downstream NG-sequencing DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina® platforms, we recommend NEBNext® Ultra™ II Library Prep Kit for Illumina® (New England Biolabs; Cat #E7645S/L).
Recommendations:
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 |
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.
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 October 2016
Protocol Id: 1184
! | This ! signifies an important step in the protocol regarding volume changes based on the number of CUT & RUN reactions being performed. |
!! | 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. |
NOTE: Steps for cell preparation (step 6-16) should be performed in succession at room temperature to minimize stress on the cells. To minimize DNA fragmentation, avoid vigorous vortexing and cavitation during resuspension.
NOTE: This protocol is written for the use of 100,000 cells per reaction. However, these same reaction conditions can be used for 10,000 to 250,000 cells per sample.
NOTE: The amount of digitonin recommended for cell permeabilization is in excess and should be sufficient for permeabilization of most cell lines. However, not all cell lines exhibit the same sensitivity to digitonin. Before you begin your experiment, it is recommended that you test your specific cell line by following the protocol provided in Appendix A. Digitonin treatment should result in permeabilization of >90% of the cell population.
! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.
NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.
NOTE: Avoid vortexing of the Concanavalin A Magnetic Bead suspension as repeated vortexing may displace the Concanavalin A from the beads.
NOTE: To avoid loss of beads, remove liquid using a pipetman. Do not aspirate using a vacuum.
NOTE: The input sample will be incubated at 55°C later in the protocol, so it is recommended to use a safe-lock 1.5 ml tube to reduce evaporation during the incubation.
NOTE: Concanavalin A Magnetic Beads may clump or stick to the sides of the tube. Beads can be resuspended by pipetting up and down.
NOTE: The amount of antibody required for CUT&RUN varies and should be determined by the user. For the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb, add 2 µl of antibody to the sample. For the negative control Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, add 5 µl to the sample. We strongly recommend using the negative control antibody and NOT a no-antibody control, because the latter results in high levels of non-specific MNase digestion and high background signal. We recommend using the input sample for comparison with both qPCR and NG-seq analysis.
! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.
NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.
! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.
NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.
NOTE: Digestion should be performed in a 4°C cooling block or refrigerator. The temperature of ice can get as low as 0°C, which can limit digestion and decrease signal.
NOTE: This incubation step can be increased to 30 min.
! All buffer volumes should be increased proportionally based on the number of input samples being prepared.
NOTE: Sonication conditions may need to be determined empirically by testing different sonicator power settings and/or durations of sonication, following the protocol in Appendix B. Optimal sonication conditions will generate chromatin fragments ranging in size from 100-600 bp. Sonication for 5 sets of 15-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe sufficiently fragments the input chromatin.
DNA can be purified from input and enriched chromatin samples using DNA spin columns, as described in Section A, or phenol/chloroform extraction followed by ethanol precipitation as described in Section B. Purification using DNA spin columns is simple and fast, providing good recovery of DNA fragments above 35 bp (Figure 7A, Lane 2). Phenol/chloroform extraction followed by ethanol precipitation is more difficult, but provides better recovery of DNA fragments below 35 bp (Figure 7A, Lane 3); however, as shown in Figure 7B, the majority of DNA fragments generated in the CUT&RUN assay are larger than 35 bp. Therefore, DNA spin columns provide a quick and simple method for purification of > 98% of the total CUT&RUN DNA fragments.
Purified DNA can be quantified prior to NG-seq analysis using a picogreen-based DNA quantification assay. For CUT&RUN reactions containing 100,000 cells, the expected DNA yield for a CUT&RUN reaction ranges from 0.5 to 10 ng per reaction for transcription factors and cofactors, and 1 to 20 ng per reaction for histone modifications.
FIGURE 7. Comparison of DNA purification using spin columns or phenol/chloroform extraction followed by ethanol precipitation. (A) A low range DNA ladder mix (lane 1, unpurified) was purified using either DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209 (lane 2) or phenol/chloroform extraction followed by ethanol precipitation (lane 3) and separated by electrophoresis on a 4% agarose gel. As shown, phenol/chloroform followed by ethanol precipitation efficiently recovers all DNA fragment sizes, while DNA spin columns recover DNA fragments ≥ 35 bp. (B) DNA was purified using phenol/chloroform extraction followed by ethanol precipitation from a CUT&RUN assay performed using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569. The size of the DNA fragments in the library was analyzed using a Bioanalyzer (Agilent Technologies). The adaptor and barcode sequences added to the library during construction account for 140 bp in fragment length. Therefore, starting 35 bp DNA fragments would be 175 bp in length after library preparation (indicated with blue vertical line in figure). As shown, less than 2% of the total CUT&RUN enriched DNA fragments are less than 175 bp (starting length of 35 bp), suggesting that DNA purification spin columns are sufficient for capture of > 98% of the total CUT&RUN DNA fragments.
NOTE: DNA can be purified from input and enriched chromatin samples using the Cell Signaling® DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209 (not included in this kit) and the modified protocol below. Steps 1 through 5 are modified to reflect the requirement for adding 5 volumes (1.5 ml) of DNA Binding Buffer to the 300 µl of input and enriched chromatin samples.
NOTE: 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.
NOTE: The following reagents are required for the phenol/chloroform extraction and ethanol precipitation and are not included in this kit: phenol/chloroform/isoamyl alcohol (25:24:1), chloroform/isoamyl alcohol (24:1), 3M Sodium Acetate (pH 5.2), 20mg/ml glycogen, 100% ethanol, 70% ethanol, and 1X TE buffer or nuclease-free water.
NOTE: If sample normalization is performed, only the CUT&RUN samples are to be analyzed using the Sample Normalization Primer Set. The input DNA does not contain the Normalization Spike-In DNA.
Reagent | Volume for 1 PCR Reaction (18 µl) |
---|---|
Nuclease-free H2O #12931 | 6 µl |
5 µM Primers | 2 µl |
SimpleChIP® Universal qPCR Master Mix #88989 | 10 µl |
a. | Initial Denaturation | 95°C for 3 min |
b. | Denature | 95°C for 15 sec |
c. | Anneal and Extension | 60°C for 60 sec |
d. | Repeat steps b and c for a total of 40 cycles. |
C[T] value of Sample Normalization Primer Set | **Normalization Factor for qPCR | Signal Before Normalization (% Input Calc'd from Step 5) | Signal After Normalization | |
Sample 1 | 23.31 | 2(23.31-23.31)=1.00 | 24.4% | 24.4%/1.00=24.4% |
Sample 2 | 24.24 | 2(23.31-24.24)=0.52 | 12.0% | 12.0%/0.52=23.1% |
Sample 3 | 25.08 | 2(23.31-25.08)=0.29 | 6.28% | 6.28%/0.29=21.7% |
Sample 4 | 26.30 | 2(23.31-26.30)=0.13 | 2.72% | 2.72%/0.13=20.9% |
**Normalization Factor for qPCR = 2(C[T] Selected Sample - C[T] the Other Sample)
FIGURE 8. Normalization of CUT&RUN signals using spike in DNA for qPCR analysis. CUT&RUN was performed with a decreasing number of HCT116 cells and either Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (upper panels) or Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499 (lower panels). Enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human β-Actin Promoter Primers #13653, SimpleChIP® Human Β-Actin 3' UTR Primers #13669, and SimpleChIP® Human MyoD1 Exon 1 Primers #4490. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin for 100,000 cells. Non-normalized enrichments are depicted in the left panels. The Sample Normalization Spike-In DNA was added into each reaction proportionally to the starting cell number. Based on the difference of qPCR signals from spike in DNA in each sample, CUT&RUN signals were normalized to the sample containing 100,000 cells. Normalized enrichments are depicted in the right panels.
The immuno-enriched DNA samples prepared with this kit are directly compatible with NG-seq. For downstream NG-seq DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina® platforms, we recommend using the SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 with SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® #29580 or #47538.
The Number of Unique Reads Aligned to Yeast | Normalization Factor for NGS | The Number of Unique Reads Aligned to Test Reference Genome Before Normalization | The Number of Unique Reads Aligned to Test Reference Genome After Normalization | |
Sample 1 | 219,275 | 219,275/219,275 = 1.00 | 5,077,747 | 5,077,747 X 1.00 = 5,077,747 |
Sample 2 | 411,915 | 219,275/411,915 = 0.53 | 9,896,671 | 9,896,671 X 0.53 = 5,268,306 |
Sample 3 | 816,235 | 219,275/816,235 = 0.27 | 17,842,773 | 17,842,773 X 0.27 = 4,793,320 |
Sample 4 | 1,120,826 | 219,275/1,120,826 = 0.20 | 23,836,679 | 23,836,679 X 0.20 = 4,663,339 |
Normalization Factor for NGS = the number of unique yeast reads from Selected Sample / the number of unique yeast reads from the other sample
In the CUT&RUN protocol, the addition of digitonin to the buffers facilitates the permeabilization of cell membranes and entry of the primary antibody and pAG-MNase enzyme into the cells and nuclei. Therefore, having an adequate amount of digitonin in the buffers is critical to the success of antibody and enzyme binding and digestion of targeted genomic loci. Different cell lines show differing sensitivities to digitonin cell permeabilization. While the amount of digitonin recommended in this protocol should be sufficient for permeabilization of most cell lines, we recommend an initial test of your specific cell line. We have found that the addition of excess digitonin is not deleterious to the assay, so there is no need to perform a concentration curve. Rather, a quick test to determine if the recommended amount of digitonin works for your cell line is sufficient.
NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.
Sonication of the input DNA sample is recommended because only fragmented genomic DNA (<10 kb) can be purified using DNA purification spin columns. Additionally, the fragmented genomic DNA (<1kb) may be used as the negative control in NG-seq analysis. Sonication should be optimized so that the input DNA is 100-600 bp in length.
We recommend using the input sample for NG-seq because it provides a convenient and unbiased representation of the cell genome. While the IgG sample can also be used as a negative control for NG-seq, it may show enrichment of specific regions of the genome due to non-specific binding. Unfragmented input DNA can be used for qPCR analysis. However, unfragmented DNA must be purified using phenol/chloroform extraction followed by ethanol precipitation.
! All buffer volumes should be increased proportionally based on the number of input samples being prepared.
NOTE: Samples will be incubated at 55°C in Step 9, so it is recommended to use a safe-lock 1.5 ml tube to reduce evaporation during the incubation.
Problem | Possible Causes | Recommendation |
---|---|---|
1. Concanavalin A beads clump during the experiment. | Bead clumping is normal and is not usually deleterious to the assay. | Resuspend clumped beads by gently pipetting up and down. |
Room temperature incubation of beads and cells is too long. | Activate Concanavalin A beads at 4°C and incubate with cells no longer than 5 min (Section I, Step 14). | |
Cells are lysing during preparation. | Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress (Section I, Steps 7-16). | |
Digitonin concentration may be too high. | Some cells may be more sensitive to digitonin and lyse at higher concentrations. Reduce the amount of digitonin in the assay, but be sure to confirm the amount used is sufficient for cell permeabilization (see APPENDIX A). | |
2. No DNA is detected in the purified DNA samples using a picogreen-based DNA quantification assay. | This is typical when starting with low cell numbers (<10,000 cells), but DNA should be detectable when starting with the recommended 100,000 cells. | Be sure to use a picogreen-based DNA quantification assay. Purified DNA is not typically detectable using a NanoDrop, Bioanalyzer® or Tapestation®. |
Cell count is off, cells are lost or lysing during preparation. | Starting cell culture should be 60-90% confluent and look healthy (> 90% live cells). | |
Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress. | ||
Wash all cells in one vial to minimize cell loss (Section I, Steps 7-16). | ||
Digitonin is not effectively permeabilizing the cells. | Be sure to store Digitonin Solution at -20°C when not in use, as it is unstable when stored above -20°C. | |
Be sure to test and confirm that the amount of digitonin used is sufficient to permeabilize your specific cell line (see APPENDIX A). | ||
pAG-MNase enzyme is not working properly in the assay. | The pAG-MNase is highly stable and should maintain activity for a long time when stored properly. | |
The pAG-MNase requires Ca2+ divalent cations for activity. Be sure to add calcium chloride for activation of the enzyme (Section III, Step 8). | ||
Be sure to digest for 30 min to allow the enzyme to sufficiently digest the chromatin (Section III, Step 9). | ||
Not enough antibody is added to the reaction or antibody does not work in the CUT&RUN assay. | Not all antibodies work in CUT&RUN. If possible, use a CUT&RUN validated antibody. Alternatively, some ChIP- and IF-validated antibodies also work for CUT&RUN. | |
Be sure to include the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb to show your assay is working. | ||
3. No signal in qPCR or NG-seq analysis. | See possible causes for problem #2. | See recommendations problem #2. |
Digestion conditions may be too cold. | We have found that performing the digestion on ice (0°C) can significantly decrease the recovery of targeted chromatin fragments, resulting in decreased qPCR and NG-seq signals. Please be sure to perform the digestion at 4°C on a cooling block or in a refrigerator. | |
Not enough DNA added to the qPCR reaction. | Add more DNA to the PCR reaction or increase the number of amplification cycles. | |
Not enough DNA added to the NG-seq DNA library preparation. | Be sure to quantify the purified DNA using a picogreen-based DNA quantification assay and use the recommended amount of starting DNA and PCR-amplification cycles (see Section VII). | |
PCR-amplified region may span a nucleosome-free region. | DNA fragments generated in the CUT&RUN assay are typically smaller than DNA fragments generated in the ChIP assay. Therefore, it is critical to design primers to generate amplicons no longer than 60 to 80 bp. | |
4. High background signal in qPCR or NG-seq analysis. | Genomic DNA has become highly fragmented due to harsh treatment of samples. | Always use the Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 negative control antibody to determine background signal in the CUT&RUN assay. |
To minimize DNA fragmentation, avoid vigorous vortexing and introduction of bubbles during resuspension of cells. | ||
Genomic DNA has become highly fragmented due to cell stress and lysis. | Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress. Wash all cells in one vial to minimize cell loss (Section I, Steps 7-16). | |
Digestion conditions may be too warm. | Digestion should be performed at 4°C in a cooling block or refrigerator. Digestion at higher temperatures can significantly increase background signal. | |
Make sure to pre-cool samples and calcium chloride on ice prior to initiating the digest. | ||
Large non-specific genomic DNA can also diffuse into the supernatant and contaminate the smaller fragments released by targeted digestion. | Do not incubate samples at 37°C for > 10 min and do not shake samples during incubation (Section III, Step 11). Ten minutes is sufficient for diffusion of digested fragments into the supernatant. | |
Large genomic DNA fragments can be removed by size-selection using AMPure® XP Beads or SPRIselect® Reagent Kit prior to qPCR analysis. | ||
For NG-seq analysis, shorten the PCR amplification time (10-15 sec) during library construction to exclude amplification of large DNA fragments. | ||
Too much antibody is used in the assay, resulting in non-specific binding and digestion. | If possible, be sure to use a CUT&RUN validated antibody at the recommended dilution. If not, ChIP-validated and IF-validated antibodies often work at their ChIP- and IF-recommended dilutions. You may need to titrate your antibody in the assay. |
posted October 2019
revised January 2021
Protocol Id: 1884
DNMT3A (D2H4B) Rabbit mAb recognizes endogenous levels of total DNMT3A protein. This antibody detects multiple isoforms of DNMT3A, including isoform 1 and isoform 2. The antibody does not cross-react with DNMT3B or other DNMT proteins.
Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro233 of human DNMT3A protein.
Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2 and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication. DNMT3A and DNMT3B are strongly expressed in embryonic stem cells with reduced expression in adult somatic tissues. DNMT3A and DNMT3B function as de novo methyltransferases that methylate previously unmethylated regions of DNA. DNMT2 is expressed at low levels in adult somatic tissues and its inactivation affects neither de novo nor maintenance DNA methylation. DNMT1, DNMT3A and DNMT3B together form a protein complex that interacts with histone deacetylases (HDAC1, HDAC2, Sin3A), transcriptional repressor proteins (RB, TAZ-1) and heterochromatin proteins (HP1, SUV39H1), to maintain proper levels of DNA methylation and facilitate gene silencing (3-8). Improper DNA methylation contributes to diseased states such as cancer (1,2). Hypermethylation of promoter CpG islands within tumor suppressor genes correlates with gene silencing and the development of cancer. In addition, hypomethylation of bulk genomic DNA correlates with and may contribute to the onset of cancer. DNMT1, DNMT3A and DNMT3B are over-expressed in many cancers, including acute and chronic myelogenous leukemias, in addition to colon, breast and stomach carcinomas (9-12).
Explore pathways + proteins related to this product.
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