Cat. # | Size | Price | Inventory |
---|---|---|---|
72364T | 20 µl | ||
72364S | 100 µl |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | 65, 75 |
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 × 10^6 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
IHC Leica Bond | 1:200 - 1:800 |
Immunohistochemistry (Paraffin) | 1:400 - 1:1600 |
Chromatin IP | 1:50 |
Chromatin IP-seq | 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 a carrier-free (BSA and azide free) version of this product see product #92761.
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 utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.
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.
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.
Proceed to one of the following specific set of steps.
NOTE: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 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) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.
When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.
posted December 2008
revised October 2021
Protocol Id: 409
NOTE: Please see product datasheet or product webpage for appropriate antibody dilution^.
Step | Reagents | Time/Temperature | |
---|---|---|---|
1 | Dewax | BOND™ Dewax Solution, 100% Alcohol, BOND™ Wash Solution | Pre-programmed Leica® BOND™ |
2 | Antigen Retrieval | BOND™ Epitope Retrieval ER2 Solution | 20 min., 100˚C | Protocol: HIER 20 min with ER2 |
3 | Peroxide Block | Refine Detection Kit Peroxide Block* | 5 min. |
WASH | BOND™ Wash Solution | 3x 0:00 min. | |
4 | Protein Block (optional) | #5425 NGS or #15019 Animal-Free Blocking Solution | 20 min. |
5 | Primary Antibody^ | Dilute in #8112 SignalStain® Antibody Diluent | 30 min. |
WASH | BOND™ Wash Solution | 3x 2:00 min. | |
NA | Post Primary Mouse Linker | Refine Detection Kit Post Primary* | Not Applied |
6 | Secondary Detection | Refine Detection Kit Polymer* | 10 min. |
WASH | BOND™ Wash Solution/Deionized Water | Custom (see below) | |
7a | Visualization | Refine Detection Kit Mixed DAB Refine* | 0:00 min. |
7b | Visualization | Refine Detection Kit Mixed DAB Refine* | 10 min. |
WASH | Deionized Water | 3x 0:00 min. | |
8 | Counterstain | Refine Detection Kit Hematoxylin* | 5 min. |
WASH | Deionized Water | 0:00 min. | |
WASH | BOND™ Wash Solution | 0:00 min. | |
WASH | Deionized Water | 0:00 min. | |
9 | Dehydration (Offline): | ||
Incubate sections in 95% ethanol two times for 10 seconds each. | |||
Repeat in 100% ethanol, incubating sections two times for 10 seconds each. | |||
Repeat in xylene, incubating sections two times for 10 seconds each. | |||
10 | Mount sections with coverslips and #84583 SignalStain® Mounting Medium | ||
Optional Custom wash: | BOND™ Wash Solution | 2:00 | |
BOND™ Wash Solution | Dispenser Type: OPEN 0:00 | ||
BOND™ Wash Solution | 2:00 | ||
BOND™ Wash Solution | Dispenser Type: OPEN 0:00 | ||
BOND™ Wash Solution | 0:00 | ||
Deionized Water | 0:00 |
*Reagent included in BOND™ Polymer Refine Detection Kit (Catalog No: DS9800)
LEICA® is a registered trademark of Leica Microsystems IR GmbH.
BOND™ is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.
posted August 2018
revised September 2018
Protocol Id: 1444
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
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: 283
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 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:
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 April 2022
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 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:
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 April 2022
Protocol Id: 1184
Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the fusion site of human SS18-SSX protein.
SS18 is a protein that has been shown to be a part of the SWI/SNF complex (1, 2). The SS18-SSX fusion proteins are a result of in-frame fusions that fuse the SS18 gene on chromosome 18 with X chromosome genes SSX1, SSX2, and to a lesser extent SSX4 (3). Human synovial sarcoma (SS) accounts for 8-10% of all soft tissue malignancies and 95% of these malignancies express the recurrent translocation of the SS18 gene on chromosome 18 (3). The N-terminal SNH domain (SYT N-terminal homology domain) of the SS18 protein interacts with SWI/SNF chromatin remodeling complexes via the N terminal region of BRM and BRG1 subunits (4). Studies of the SS18-SSX fusion in SS suggest that endogenous SS18 competes with the mutant SS18-SSX fusion for occupancy in the SWI/SNF complexes resulting in the displacement of SNF5 (BAF47) subunit. Displacement of the SNF5 subunit results in altered function of the SWI/SNF complex that leads to deregulated expression of genes such as Sox2 in SS (3).
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