Description
General description
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 (Lys27) set includes the Dimethyl-Histone H3 (Lys27) antibody, a negative control rabbit supernatant, and qPCR primers which amplify a 110 bp region of human β-globin promoter. The Dimethyl-Histone H3 (Lys27) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Dimethyl-Histone H3 (Lys27)-associated chromatin.
Histones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin. The four core histones, H2A, H2B, H3, and H4, assemble into an octamer (2 molecules of each). Subsequently, 146 base pairs of DNA are wrapped around the octamer, forming a nucleosome. Histones are modified post-translationally by the actions of enzymes in both the nucleus and cytoplasm. These modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. These modifications can alter local chromatin architecture, or recruit trans-acting factors that recognize specific histone modifications (the "histone code" hypothesis). The modifications occur predominantly on the N-terminal and C-terminal tails that extend beyond the nucleosome core particle. Histone H3 is methylated at Lys27 by EZH2, and and overexpression of EZH2 has been associated with both breast and prostate cancers. Methylation of H3K27 is involved in X chroosome inactivation, imprinting, circadian rhythms, and stem cell maintenance. H3K27me2 is a marker of classical heterochromatin.
~17 kDa
Immunogen
Epitope: Dimethyl Lys27
Peptide containing the sequence (ARme2KSA) in which me2 corresponds to dimethyl lysine at residue 27 of human histone H3.
Application
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using either 2 µL of Negative Control Supernatant or 2 µL of Anti-dimethyl-Histone H3 (Lys27) and the Magna ChIP® A Kit (Cat. # 17-610).
Successful immunoprecipitation of dimethyl-Histone H3 (Lys27) associated DNA fragments was verified by qPCR using ChIP Primers, human β-globin as a positive locus, and GAPDH promoter primers as a negative locus (Please see figures). Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Western Blot Analysis:
Representative lot data.
A 1:1000-1:5000 dilution of a previous lot detected dimethyl-Histone H3 in acid extracted proteins from HeLa cells, but did not detect unmethylated recombinant Histone H3 (Catalog # 14-494).
Recombinant Histone H3 (lane 1) and HeLa cell acid precipitate (lane 2) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-dimethyl-Histone H3 (Lys27) (1:1000 dilution).
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates dimethyl-histone H3 (~17 kDa) (Please see figures).
Peptide Inhibition Assay (PIA):
Representative lot data.
0.5-2 μM of histone H3 peptides containing dimethyl-Lys27 abolished detection of histone H3 by anti-dimethyl-Histone H3 (Lys27) (1:1000 dilution) in immunoblots of acid extracted proteins from HeLa cells.
Acid extracted proteins from HeLa cells were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-dimethyl-Histone H3 (Lys27) (lane 1) or anti-dimethyl-Histone H3 (Lys27) preabsorbed with 0.5 mM of histone H3 peptides containing the following modifications:
Lane 2: dimethyl-lysine 23
Lane 3: dimethyl-lysine 27
Lane 4: dimethyl-lysine 9
A 1:1000 dilution of the primary antibody was used.
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates dimethyl-histone H3 (~17 kDa) (Please see figures).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Histones
This ChIPAb+ Dimethyl-Histone H3 (Lys27) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Biochem/physiol Actions
Broad species cross-reactivity expected, based on sequence identity in most species.
This antibody recognizes Histone H3 dimethylated on Lys27.
Packaging
25 assays per set. Recommended use: ~2 μL of antibody per chromatin immunoprecipitation (dependent upon biological context).
Physical form
Anti-Dimethyl-Histone H3 (Lys27) (rabbit monoclonal). One vial containing 50 µL of cultured supernantant in 0.05% sodium azide. Store at -20°C.
Negative Control Supernatant. One vial containing 100 µL of rabbit cultured supernatant in 0.05% sodium azide. Store at -20°C.
ChIP Primers, human β-globin. One vial containing 75 μL of 5 μM of each primer specific for the human β-globin promoter. Store at -20°C.
FOR: AGG ACA GGT ACG GCT GTC ATC
REV: TTT ATG CCC AGC CCT GGC TC
Unpurified supernatant
Preparation Note
1 year at -20°C from date of shipment
Handling Recommendations: Upon receipt, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/ thaw cycles, which may damage IgG and affect product performance.
Analysis Note
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using either 2 µL of Negative Control Supernatant or 2 µL of Anti-dimethyl-Histone H3 (Lys27) and the Magna ChIP® A Kit (Cat. # 17-610).
Successful immunoprecipitation of dimethyl-Histone H3 (Lys27)-associated DNA fragments was verified by qPCR using ChIP Primers, human β-globin (Please see figures).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Control
Includes negative control rabbit supernatant and primers specific for human β-globin promoter.
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.
