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Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (Sepharose Bead Conjugate) #4232
Immunoprecipitation of tri-methyl-histone H3 (Lys27) from HeLa cell extracts using Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (Sepharose Bead Conjugate) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose Bead Conjugate) #3423. The western blot was probed using Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733 and Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 as a secondary antibody.Learn more about how we got this image
Gallery: Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (Sepharose Bead Conjugate) #4232
Immunoprecipitation for Analysis by Western Blotting
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
- 20X Phosphate Buffered Saline (PBS): (#9808).
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
- 3X SDS Sample Buffer: (#7722) 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue
- 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
- ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.
B. Preparing Cell Lysates
- Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
- To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
- Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer to each plate (10 cm) and incubate the plates on ice for 5 minutes.
- Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
- Sonicate samples on ice three times for 5 seconds each.
- Microcentrifuge for 10 minutes at 4°C, 14,000 x g, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.
- Take 200 μl cell lysate and add 20 μl of the immobilized antibody, incubate with rotation overnight at 4°C.
- Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice during washes.
- Proceed to sample analysis by western blotting or kinase activity (section D).
D. Sample Analysis
Proceed to one of the following specific set of steps.
For Analysis by Western Immunoblotting
- Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
- Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
- Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
- Analyze sample by western blot (see Western Immunoblotting Protocol).
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
For Analysis by Kinase Assay
- Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
- Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
- Incubate for 30 min at 30°C.
- Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
- Transfer supernatant containing phosphorylated substrate to another tube.
- Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
- Load the sample (15–30 µl) on SDS-PAGE (4–20%).
posted December 2007
protocol id: 27
Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (Sepharose Bead Conjugate) recognizes endogenous levels of histone H3 only when tri-methylated on Lys27. The antibody does not cross-react with non-methylated, mono-methylated, or di-methylated Lys27. In addition, the antibody does not cross-react with mono-methylated, di-methylated, or tri-methylated histone H3 at Lys4, Lys9, Lys36, or Histone H4 at Lys20.Species Reactivity: Human, Mouse, Rat, Monkey Species predicted to react based on 100% sequence homology: Xenopus, Zebrafish
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys27 is tri-methylated.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated sepharose beads. Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (Sepharose Bead Conjugate) is useful for the immunoprecipitation of tri-methyl-histone H3 (Lys27). The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733.
The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1). Histone methylation is a major determinant for the formation of active and inactive regions of the genome and is crucial for the proper programming of the genome during development (2,3). Arginine methylation of histones H3 (Arg2, 17, 26) and H4 (Arg3) promotes transcriptional activation and is mediated by a family of protein arginine methyltransferases (PRMTs), including the co-activators PRMT1 and CARM1 (PRMT4) (4). In contrast, a more diverse set of histone lysine methyltransferases has been identified, all but one of which contain a conserved catalytic SET domain originally identified in the Drosophila Su(var)3-9, Enhancer of zeste, and Trithorax proteins. Lysine methylation occurs primarily on histones H3 (Lys4, 9, 27, 36, 79) and H4 (Lys20) and has been implicated in both transcriptional activation and silencing (4). Methylation of these lysine residues coordinates the recruitment of chromatin modifying enzymes containing methyl-lysine binding modules such as chromodomains (HP1, PRC1), PHD fingers (BPTF, ING2), tudor domains (53BP1), and WD-40 domains (WDR5) (5-8). The discovery of histone demethylases such as PADI4, LSD1, JMJD1, JMJD2, and JHDM1 has shown that methylation is a reversible epigenetic marker (9).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 5,675,063.