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4232S 400 µl (40 immunoprecipitations)

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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous 17 Rabbit IgG
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Immunoprecipitation

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.

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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.

  1. 20X Phosphate Buffered Saline (PBS): (#9808).
  2. 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*.

  1. 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
  2. 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.
  3. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
  3. 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.
  4. Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate samples on ice three times for 5 seconds each.
  6. 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.

C. Immunoprecipitation

  1. Take 200 μl cell lysate and add 10 μl of the immobilized antibody, incubate with rotation overnight at 4°C.
  2. Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice during washes.
  3. 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

  1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (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

  1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15–30 µl) on SDS-PAGE (4–20%).

posted December 2007

protocol id: 27

Product Usage Information

Application Dilutions
Immunoprecipitation 1:20

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.

Specificity / Sensitivity

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

Source / Purification

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.

Product Description

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).


1.  Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.

2.  Kubicek, S. et al. (2006) Ernst Schering Res Found Workshop , 1-27.

3.  Lin, W. and Dent, S.Y. (2006) Curr Opin Genet Dev 16, 137-42.

4.  Lee, D.Y. et al. (2005) Endocr Rev 26, 147-70.

5.  Daniel, J.A. et al. (2005) Cell Cycle 4, 919-26.

6.  Shi, X. et al. (2006) Nature 442, 96-9.

7.  Wysocka, J. et al. (2006) Nature 442, 86-90.

8.  Wysocka, J. et al. (2005) Cell 121, 859-72.

9.  Trojer, P. and Reinberg, D. (2006) Cell 125, 213-7.


Entrez-Gene Id 8350
Swiss-Prot Acc. P68431


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.