Histone H3K9me3 antibody (pAb)

RRID: AB_2532132
Catalog No: 39161 Format: 100 µl $455 Buy
Catalog No: 39062 Format: 50 µl $265 Buy
Catalog No: 39162 Format: 10 µl $110 Buy

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Antibody Type:
Polyclonal
Isotype:
Serum
Purification:
None
Host:
Rabbit
Molecular Weight:
17 kDa
Reactivity:
Fission Yeast, Human, Wide Range Predicted

Applications

CUT&Tag Validated ChIP Validated ChIP-Seq Validated Western Blot Validated Immunofluorescence Validated Dot Blot Validated Immunohistochemistry Validated Immunocytochemistry Validated

Application Notes

Applications Validated by Active Motif:
ChIP: 2 - 10 µl per ChIP
ChIP-Seq: 10 µl each
ICC/IF: 1:500 - 1:1,000 dilution
WB*: 1:1,000 - 1:5,000 dilution
CUT&Tag: 1 µl per 50 µl reaction

*This antibody has been validated for CUT&Tag using Active Motif's CUT&Tag-IT™ Assay Kit, Catalog No. 53160.

*Note: many chromatin-bound proteins are not soluble in a low salt nuclear extract and fractionate to the pellet. Therefore, we recommend a High Salt / Sonication Protocol when preparing nuclear extracts for Western blot.

Published Applications

The following applications have been published using this antibody. Unless noted above, Active Motif may not have validated the antibody for use in these applications:

ChIP, ChIP-Seq
Native ChIP
IF
WB

View publications that use Active Motif products & services here. Enter the product number(s) to see publications & applications that use this antibody.

Immunogen

This Histone H3 trimethyl Lys9 antibody was raised against a peptide including trimethyl-lysine 9 of histone H3.

Buffer

Rabbit serum containing 30% glycerol and 0.035% sodium azide. Sodium azide is highly toxic. For your convenience, an IgG version (Catalog No. 39765) of this antibody that was purified by Protein A Chromatography is also available.

References

Histone H3K9me3 antibody (pAb) - 100 µg (Cat. No. 39161)

 
 
 

Histone H3K9me3 antibody (pAb) - 10 µg (Cat. No. 39162)

 
 
 

 

ChIP-Seq of Histone H3K9me3 pAb
ChIP was performed using the ChIP-IT® High Sensitivity Kit (Cat. No. 53040) with 15 µg of chromatin from D425 human Medulloblastoma cells and 10 µl of Histone H3K9me3 antibody (pAb). ChIP DNA was sequenced on the Illumina HiSeq and 16 million sequence tags were mapped to identify H3K9me3 binding sites. H3K9me3 binds broadly across the genome. The image shows two different regions. The left box shows many binding sites across a broad region on chromosome 1. The right box is zoomed in to show a dispersed binding pattern across the RHOT1 gene.

Histone H3 trimethyl Lys9 antibody tested by immunofluorescence.
HeLa cells stained at 2 µg/ml with Histone H3 trimethyl Lys9 antibody. Top panel: Histone H3 trimethyl Lys9 antibody. Middle panel: DAPI. Bottom panel: merge.

Histone H3K9me3 antibody (pAb) tested by Western blot.
HeLa cell nuclear extract (10 µg per lane) probed with Histone H3K9me3 antibody (pAb) at a dilution of 1:2,000.

Histone H3 trimethyl Lys9 antibody specificity tested by peptide array analysis.
Peptide array analysis was used to confirm the specificity of this antibody for its intended modification. Histone H3 trimethyl Lys9 antibody was applied at a dilution of 1:5,000 to Active Motif's MODified™ Histone Peptide Array (Catalog No. 13001). The arrays were scanned with ArrayAnalysis Software 7 and the results plotted. Specificity data is shown for the most reactive peptides.

Dot blot of Histone H3 trimethyl Lys9 antibody.
Dot blot analysis was used to confirm the specificity of H3K9me3 antibody for trimethyl Lys9 of histone H3. Recombinant methylated peptides corresponding to the immunogen and related sequences were spotted onto PVDF and probed with H3K9me3 at 2 µg/ml. The amount of protein (picomoles) spotted is indicated next to each row.
Top panel - Lane 1: unmodified H3K4. Lane 2: H3K4me1. Lane 3: H3K4me2. Lane 4: H3K4me3. Lane 5: unmodified H3K9. Lane 6: H3K9me1. Lane 7: H3K9me2. Lane 8: H3K9me3. Lane 9: unmodified H3K79. Lane 10: H3K79me1. Lane 11: H3K79me2. Lane 12: H3K79me3. Bottom panel - Lane 1: unmodified H3K23. Lane 2: H3K23me1. Lane 3: H3K23me3. Lane 4: H3K23me3. Lane 5: unmodified H3K27. Lane 6: H3K27me1. Lane 7: H3K27me2. Lane 8: H3K27me3. Lane 9: unmodified H3K36. Lane 10: H3K36me1. Lane 11: H3K36me2. Lane 12: H3K36me3.

Background

Histone H3 is one of the core components of the nucleosome. The nucleosome is the smallest subunit of chromatin and consists of 147 base pairs of DNA wrapped around an octamer of core histone proteins (two each of Histone H2A, Histone H2B, Histone H3 and Histone H4). Histone H1 is a linker histone, present at the interface between the nucleosome core and DNA entry/exit points. Histone H1 is responsible for establishing higher-order chromatin structure. Chromatin is subject to a variety of chemical modifications, including post-translational modifications of the histone proteins and the methylation of cytosine residues in the DNA. Reported histone modifications include acetylation, methylation, phosphorylation, ubiquitylation, glycosylation, ADP-ribosylation, carbonylation and SUMOylation; these modifications play a major role in regulating gene expression.

The methylation of histones can occur on two different residues: arginine or lysine. Histone methylation can be associated with transcriptional activation or repression, depending on the methylated residue. Lysine 9 of histone H3 can be mono-, di- or trimethylated by different histone methyltransferases (HMTs) such as SuvH39H1 or G9a. This methylated lysine can be demethylated by histone demethylases as JMJD1A, LSD1 or JMJD2C. Methylation of this residue is mainly associated with transcriptional repression.

Positive Control

Active Motif's HeLa acid extract (Catalog No. 36200) can be used as a positive control for Histone H3 trimethyl Lys9 antibody.

Chromatin IP Assays

This antibody has been validated for use in ChIP and/or ChIP-Seq, and can be used with Active Motif's ChIP-IT® High Sensitivity Kit or our magnetic bead-based ChIP-IT® Express Kits. For an overview of all of our ChIP products, please visit our Chromatin IP Page to learn about ChIP products designed for use with Histone H3K9me3 antibody (pAb).

Storage

Some products may be shipped at room temperature. This will not affect their stability or performance. Avoid repeated freeze/thaw cycles by aliquoting items into single-use fractions for storage at -20°C for up to 2 years. Keep all reagents on ice when not in storage.

Guarantee

This product is guaranteed for 12 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.

Application Key

  • ChIP = Chromatin Immunoprecipitation;
  • ChIP = ChIP Sequencing;
  • CUT&RUN = Cleavage Under Targets and Release Using Nuclease;
  • CUT&Tag = Cleavage Under Targets and Tagmentation;
  • DB = Dot Blot;
  • ELISA = Enzyme-linked Immunosorbent Assay;
  • EMSA = Electrophoretic Mobility Shift Assay
  • FC = Flow Cytometry;
  • ICC = Immunocytochemistry;
  • IF = Immunofluorescence;
  • IHC = Immunohistochemistry;
  • IP = Immunoprecipitation;
  • MeDIP = Methyl-DNA Immunoprecipitation;
  • WB = Western Blotting