Analysis Note

ControlIncluded negative control antibody normal rabbit serum and control primers specific for human β-globin promoter.

Application

Dimethyl-Histone H3 (Lys9) ChIP validated antibody & primer set including the ChIP-grade antibody & the specific control PCR primers used for chromatin immunoprecipitation of H3K9Me2.

Research CategoryEpigenetics & Nuclear Function

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from untreated HeLa cells (1 X 10⁶ cell equivalents) was subjected to chromatin Immunoprecipitation using 4 µL of either a normal rabbit antiserum or Antidimethyl-Histone H3 (Lys9) serum and the Magna ChIP A (Cat. #17-610) Kit (Please see figures). Successful Immunoprecipitation of dimethyl-histone H3 (Lys9) associated DNA fragments was verified by qPCR using β-globin ChIP Primers flanking the human β-globin promoter or primers amplifying the promoter of human GAPDH, which is transcriptionally inactive in HeLa cells. Percent Input relative to standard curves for each qPCR primer set are shown.
Please refer to the EZ-Magna A ChIP™ (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.

Western blot analysis and peptide inhibition:
HeLa Acid extract were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-dimethyl-Histone H3 (Lys9) (1:500, Lane 1) or preincubated with 0.4 µM Histone H3 peptide with following modifications:
Lane 2: Linear non-modified
Lane 3: Branched non-modified
Lane 4: Branched trimethyl
Lane 5: Linear trimethyl
Lane 6: Branched dimethyl
Lane 7: Linear dimethyl
Lane 8: Branched monomethyl
Lane 9: Linear monomethyl
Proteins were visualized using a goat anti–rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.

Research Sub CategoryChromatin Biology

Components

Anti-Dimethyl-Histone H3 (Lys9) (rabbit serum), 1 vialNegative ChIP Control serum, 1 vialChIP Primers β-globin , 1 vial

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

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.

All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction.
The ChIPAb+ Dimethyl-Histone H3 (Lys9) set includes the anti-dimethyl-histone H3 (Lys9) antibody, a negative control antibody (normal rabbit serum), and qPCR primers which amplify a 110 bp region within the promoter of the human β-globin gene. The dimethyl-histone H3 (Lys9) and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of dimethyl-histone H3 (Lys9) associated chromatin.

Immunogen

The dimethyl-histone H3 (Lys9) rabbit serum is made against a KLH-conjugated, branched synthetic peptide containing the sequence ..Rme2KSTG.., in which me2K corresponds to dimethyl-lysine at residue 9 of human histone H3

Epitope: Dimethyl Lys9

Legal Information

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Packaging

25 assays per kit, ~4µL per chromatin immunoprecipitation

Physical form

Dimethyl-Histone H3 (Lys9) (rabbit polyclonal serum). One vial containing 100 µL of antiserum containing 0.05% sodium azide.

Normal Rabbit Serum. One vial containing 100 uL antiserum containing 0.05% sodium azide.

ChIP Primers, β-Globin. One vial containing 75 µL of 5 µM of each primer specific for human β-globin.
FOR: AGG ACA GGT ACG GCT GTC ATC
REV: TTT ATG CCC AGC CCT GGC TC

Quality

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from untreated HeLa cells (1 X 10⁶ cell equivalents) was subjected to chromatin immunoprecipitation using 4 µL of either a normal rabbit antiserum or 4 µL Anti-Dimethyl-Histone H3 (Lys9) serum and the Magna ChIP A (Cat. #17-610) Kit.
Successful immunoprecipitation of dimethyl histone H3 (Lys9) associated DNA fragments was verified by qPCR using control ChIP Primers flanking the β-globin human promoter (Please see figures).

Specificity

Dimethyl-Histone H3 (Lys9)

Storage and Stability

Stable for 1 year at -20°C from date of receipt

Target description

17 kDa