Revision 6
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, ChIP, ChIP-seq, C&R, C&T

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

11

Source/Isotype:

Rabbit IgG

UniProt ID:

#P62805

Entrez-Gene Id:

8359

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.

The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.

Application Dilution
Western Blotting 1:1000
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50
CUT&Tag 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.

Specificity / Sensitivity

Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb recognizes endogenous levels of histone H4 protein only when tri-methylated at Lys20. This antibody does not cross-react with non-methylated, mono-methylated or di-methylated histone H4 Lys20. This antibody detects a 95 kDa non-specific protein of unkown origin.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Xenopus, Bovine, Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H4 in which Lys20 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 ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN C&T: CUT&Tag

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.
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 6
#5737

Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb

Western Blotting Image 1: Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa and NIH/3T3 cells using Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb.
Chromatin Immunoprecipitation Image 1: Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb or Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb #13969, 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. H4K20me3 and H3K9me3 are known to have similar binding pattern on chromatin. The figure shows binding of both H4K20me3 and H3K9me3 across ZNF genes, known target genes of both H4K20me3 and H3K9me3.
Chromatin Immunoprecipitation Image 2: Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb or Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb #13969, 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. H4K20me3 and H3K9me3 are known to have similar binding pattern on chromatin. The figure shows binding of both H4K20me3 and H3K9me3 across chromosome 19 (upper), including ZNF genes (lower), known target genes of both H4K20me3 and H3K9me3.
Chromatin Immunoprecipitation Image 3: Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Tri-Methyl-Histone H4 (Lys20) (D84D2) 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 α Satellite Repeat Primers #4486, SimpleChIP® Human AFM Intron 1 Primers #5098, and SimpleChIP® Human GAPDH Exon 1 Primers #5516. 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 H4 (Lys20) (D84D2) Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells and Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across LRIF1, a known target gene of Tri-Methyl-Histone H4 (Lys20) (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells and Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 1 (upper), including LRIF1 (lower), a known target gene of Tri-Methyl-Histone H4 (Lys20) (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb Expand Image
CUT&RUN was performed with HeLa cells and treatment and either Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb without product number 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 LRIF1 promoter primers, human COX7C promoter primers, and human CETN3 promoter primers. 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 Tag Image 1: Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb Expand Image
CUT&Tag was performed with HeLa cells and Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across the LRIF1 gene.
CUT and Tag Image 2: Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb Expand Image
CUT&Tag was performed with HeLa cells and Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across chromosome 1 (upper), including the LRIF1 gene (lower).