ChIPAb+ Monomethyl-Histone H3 (Lys27)-ChIP Validated Antibody and Primer Set

32160702

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+ Monomethyl-Histone H3 (Lys27) set includes the anti-monomethyl-histone H3 (Lys27) antibody, a negative control antibody (normal rabbit serum), and qPCR primers which amplify a 110 base pair region within the promoter of the human β-globin gene. The monomethyl-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 monomethyl-histone H3 (Lys27)-associated chromatin.

Monomethyl-Histone H3 (Lys27)

The monomethyl-histone H3 (Lys27) antiserum is made against a synthetic 2X-branched peptide containing the sequence…ARmeKSA…in which meK corresponds to monomethyl-lysine 27 of human histone H3.

This ChIPAb+ Monomethyl-Histone H3 (Lys27) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Chromatin Immunoprecipitation:
Successful immunoprecipitation of monomethyl-histone H3 (Lys27) associated DNA fragments was verified by qPCR using control ChIP Primers flanking the human GAPDH promoter or primers amplifying the promoter of human β-globin, which is transcriptionally inactive in HeLa cells (Please see figures). Percent Input relative to standard curves for each qPCR primer set are shown, with immunoprecipitated DNA from control serum shown as (-) and monomethyl- histone H3 (Lys27) as (+).
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 was resolved by electrophoresis, transferred to nitrocellulose and probed with Anti-Monomethyl-Histone H3 (Lys27) (1:5000, Lane 1) or preincubated with 0.4 μM Histone H3 peptide with following modifications:
Lane 2: Linear non-modified
Lane 3: Branched monomethyl
Lane 4: Linear monomethyl
Lane 5: Branched dimethyl
Lane 6: Linear dimethyl
Lane 7: Branched trimethyl
Lane 8: Linear trimethyl
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
Chromatin Biology

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 Anti-Monomethyl-Histone H3 (Lys27) serum and the Magna ChIP A Kit (Cat. #17-610).
Successful immunoprecipitation of monomethyl-histone H3 (Lys27) associated DNA fragments was verified by qPCR using control ChIP Primers flanking the human β-globin promoter (Please see figures).
Please refer to the EZ-Magna A ChIP (Cat. #17-408) or EZ-ChIP (Cat. #17-371).

Anti-monomethyl-Histone H3 (Lys27) (rabbit polyclonal IgG). 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 for human β-globin promoter.
FOR: AGG ACA GGT ACG GCT GTC ATC
REV: TTT ATG CCC AGC CCT GGC TC

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

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

rabbit

100

serum

polyclonal

mouse, human

mammals

ChIPAb+
Upstate^®

ChIP: suitable
electrophoretic mobility shift assay: suitable
flow cytometry: suitable
immunofluorescence: suitable
immunohistochemistry (formalin-fixed, paraffin-embedded sections): suitable
immunoprecipitation (IP): suitable
western blot: suitable

NM_003493

Q66I33

dry ice

human ... H3F3B(3021)

25 assays per kit, ~4μL per chromatin immunoprecipitation

17 kDa

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

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.

10 - Combustible liquids