Revision 9
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IHC-P, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

17

Source/Isotype:

Rabbit IgG

UniProt ID:

#P68431

Entrez-Gene Id:

8350

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunohistochemistry (Paraffin) 1:50 - 1:200
Immunofluorescence (Immunocytochemistry) 1:25600 - 1:102400
Flow Cytometry (Fixed/Permeabilized) 1:50 - 1:200
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50

Storage

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 #53775.

Specificity / Sensitivity

Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb detects endogenous levels of histone H3 only when tri-methylated on Lys36. The antibody does not cross-react with non-methylated, mono-methylated, or di-methylated Lys36. In addition, the antibody does not cross-react with histone H3 methylated at Lys4, Lys9, Lys27 or histone H4 methylated at Lys20.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Hamster, Chicken, D. melanogaster, Xenopus, Zebrafish, Bovine

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys36 is tri-methylated.

Background

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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: 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.

Applications Key

WB: Western Blotting IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 9
#4909

Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb

Western Blotting Image 1: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb.
Immunohistochemistry Image 1: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human papillary carcinoma of the breast using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb.
Immunohistochemistry Image 2: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb.
Immunohistochemistry Image 3: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded 786-O cell pellet (left, positive) or A498 cell pellet (right, negative) using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb. Note that the A498 cell line harbors a SETD2 mutation.
Immunohistochemistry Image 4: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb in the presence of non-methyl peptide (left) or K36 tri-methyl peptide (right).
Immunohistochemistry Image 5: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Tri-Methyl-Histone H3(K36) (D5A7) XP(R) Rabbit mAb.
Immunofluorescence Image 1: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb (green) and COX IV (4D11-B3-E8) Mouse mAb #11967 (red).
Flow Cytometry Image 1: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Flow cytometric analysis of A498 cells (blue) and 786-O cells (green) using Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ACTG1/γ-Actin, a known target gene of H3K36me3 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 17 (upper) and ACTG1/γ-Actin (lower), a known target gene of H3K36me3 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human γ-Actin Promoter Primers #5037, SimpleChIP® Human γ-Actin Intron 3 Primers #5047, SimpleChIP® Human GAPDH Promoter Primers #4471, and SimpleChIP® Human GAPDH Intron 2 Primers #4478. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT and RUN Image 1: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells and Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across JTB, a known target gene of H3K36me3 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells and Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figures show binding across ATCG1 gene (upper) and JTB (lower), a known target gene of H3K36me3 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells and either Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human JTB exon 1 and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Product Image 1: Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb Expand Image
Tri-Methyl Histone H3 (Lys36) (D5A7) XP® Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to pre-coated tri-methyl histone H3 (Lys36) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the tri-methyl histone H3 (Lys36) peptide competed away binding of the antibody.