Upstream / Downstream

Explore pathways related to this product.

Our U.S. Offices Will Be Closing Early

Our U.S. offices will be closing early on November 24th at 6:00 PM EST.

To Purchase # 11960S

11960S 100 µl (50 tests) $299.00
$ 0. 00

Questions?

Find answers on our FAQs page.

ANSWERS  

Visit PhosphoSitePlus®

PTM information and tools available.

LEARN MORE

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Dm Sc Endogenous 17 Rabbit IgG
Image
Image
Image

Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (Alexa Fluor® 555 Conjugate) (red). Actin filaments were labeled with Alexa Fluor® 488 Phalloidin #8878 (green).

Learn more about how we get our images
Image
Image
Image
Page

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. 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.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

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.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

protocol id: 182

Product Usage Information

Application Dilutions
Immunofluorescence 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (Alexa Fluor® 555 Conjugate) detects endogenous levels of histone H3 when tri-methylated on Lys4. This antibody shows some cross-reactivity with histone H3 that is di-methylated on Lys4, but does not cross-react with non-methylated or mono-methylated histone H3 Lys4. In addition, the antibody does not cross-react with methylated histone H3 Lys9, Lys27, Lys36 or methylated histone H4 Lys20.


Species Reactivity: Human, Mouse, Rat, Monkey, D. melanogaster, S. cerevisiae
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 Lys4 is tri-methylated.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751.


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.
The Alexa Fluor® dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
U.S. Patent No. 5,675,063.