Description
General description
17kDa
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+ Trimethyl-Histone H3 (Lys27) set includes the Anti-Trimethyl-Histone H3 (Lys27) antibody, a negative control antibody (purified rabbit IgG), and qPCR primers which amplify a 139 bp region of human alpha-Satellite. The Trimethyl-Histone H3 (Lys27) and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Trimethyl-Histone H3 (Lys27)-associated chromatin.
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 27 of histone H3 can be mono-, di- or trimethylated (Histone H3 monomethyl Lys27, Histone H3 dimethyl Lys27 or Histone H3 trimethyl Lys27) by different histone methyltransferases such as EZH2 or NSD3. Methylation of this residue is mainly associated with transcriptional repression.
Immunogen
Epitope: Trimethyl Lys27
KLH-conjugated, synthetic 2X-branched peptide containing the sequence ...AR(me3K)SAP... in which me3K corresponds to trimethyl-lysine at residue 27 of human Histone H3.
Application
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from 2x10⁶ HeLa cells were subjected to chromatin immunoprecipitation using 4 μg purified anti-trimethyl-Histone H3 (Lys27) antibody or normal rabbit IgG and the Magna ChIP A kit (Cat. #17-610). Successful enrichment of trimethyl-Histone H3 (Lys27) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH flanking the human GAPDH promoter and primers targeting the promoter of human MyoD.
Please refer to the EZ-Magna ChIP A (Cat. #17-408) or EZChIP (Cat. #17-371) kit protocols for experimental details.
Western Blot Analysis:
Recombinant Histone H3 (Lane 2) and HeLa cell acid extracts (Lane 1) were resolved by electrophoresis, transferred to nitrocellulose and probed with a 1:5000 dilution of anti- trimethyl histone H3 Lys27 (0.4 μg/mL).
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system (Please see figures).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Histones
Trimethyl-Histone H3 (Lys27) ChIP validated antibody & primer set including the ChIP-grade antibody & the specific control PCR primers used for chromatin immunoprecipitation of H3K27Me3.
Biochem/physiol Actions
Broad species cross-reactivity expected.
trimethyl-Histone H3 (Lys27)
Packaging
25 assays per kit, ~4μL per chromatin immunoprecipitation.
Physical form
Anti-trimethyl-Histone H3 (Lys27) (rabbit polyclonal IgG). One vial containing 100 μg protein A purified IgG in 100 μL buffer containing 0.1 M Tris-Glycine, pH 7.4, 0.15M NaCl with 0.05% sodium azide. Store at -20°C.
Normal Rabbit IgG. One vial containing 125 μg purified Rabbit IgG in 125 μL storage buffer containing 0.1% sodium azide. Store at -20°C.
ChIP Primers human alpha-Satellite. One vial containing 75 μL of 5 μM of each control primer specific for human alpha-Satellite. Store at -20°C.
FOR: CTG CAC TAC CTG AAG AGG AC
REV: GAT GGT TCA ACA CTC TTA CA
Preparation Note
1 year at -20°C from date of shipment
Analysis Note
Chromatin Immunoprecipitation:
Sonicated Chromatin prepared from 3x10⁶NIH3T3 L1 cells were subjected to chromatin immunoprecipitation using 4 μg purified antibody or normal rabbit IgG and the Magna ChIP® A kit (Cat. #17-610). Successful enrichment of trimethyl-Histone H3
(Lys27) associated DNA fragments was verified by qPCR using ChIP Primers human alpha-Satellite (Please see figures).
Please refer to the EZ-Magna ChIP A (Cat. #17-408) or EZ-ChIP (Cat. #17-371) kit protocols for experimental details.
Control
1 vial containing 75μl of 5μM of each control primer specific for human alpha-Satellite. Store at -20°C.
FOR: CTG CAC TAC CTG AAG AGG AC
REV: GAT GGT TCA ACA CTC TTA CA
Other Notes
Anti-trimethyl Histone H3(Lys27) polyclonal, 1 vial
Polyclonal control rabbit serum, 1 vial
Human alpha-Satellite primer set, 1 vial
Legal Information
MAGNA CHIP is a registered trademark of Merck KGaA, Darmstadt, Germany
UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany
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.
