Cat. # | Size | Qty. | Price |
---|---|---|---|
94032S | 100 µl |
|
REACTIVITY | H Mk |
SENSITIVITY | Endogenous |
MW (kDa) | 100 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
Chromatin IP | 1:50 |
Chromatin IP-seq | 1:50 |
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, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala10 of human BRD3 protein.
Bromodomain-containing protein 3 (BRD3), also known as ORFX, is a member of the bromodomains and extra terminal (BET) family of proteins, which also includes BRD2, BRD4, and BRDT (1-3). BET family proteins contain two tandem bromodomains and an extra terminal (ET) domain, and bind acetyl lysine residues (3). BRD3 is capable of binding acetylated histone H3 Lys14 as well as acetylated histone H4 Lys5 and Lys12 to promote transcription (4). BRD3 plays a role in erythroid development by binding to GATA1 to facilitate its binding to target genes (5). Similar to BRD4, the BRD3 gene can be fused to NUT in NUT midline carcinomas (6). Investigators have found molecular inhibition of BET proteins to be effective in inducing apoptosis in various MLL-fusion driven leukemic cell lines by competing BRD3 and BRD4 from chromatin, leading to reduced expression of BCL2, Myc, and CDK6 (7).
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