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+ Monomethyl-Histone H3 (Lys9) set includes the Anti-monomethyl-Histone H3 (Lys9) antibody, a negative control antibody (purified Mouse IgG), and qPCR primers in the GAPDH coding region, amplifying a 213 base pair PCR product. The monomethyl-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 monomethyl-histone H3 (Lys9) associated chromatin.
Monomethyl-histone H3 at ~17 kDa
Immunogen
Epitope: a.a. 1-18
The monomethyl-histone H3 (Lys9) purified antibody is made against a synthetic peptide (monomethylated at Lys9) corresponding to amino acids 1-18 of Histone H3.
Application
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 106 cell equivalents per IP) was subjected to chromatin immunoprecipitation using 2 µg of either a normal mouse IgG or Anti-monomethyl-Histone H3 (Lys9) antibody and the Magna ChIP G (Cat. #17-611) Kit. Successful immunoprecipitation of monomethyl-histone H3 (Lys9) associated DNA fragments was verified by qPCR using GAPDH Coding region primers versus Control Primers directed against the GAPDH promoter region (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 G ChIP (Cat. #17-408) or EZ-ChIP (Cat. #17-371) protocol for experimental details.
Western Blot Analysis:
Representative data of previous lot. HeLa acid extract (Lane 1) was resolved by electrophoresis, transferred to PVDF membrane and probed with Anti-monomethyl Histone H3 (Lys9) (0.5 μg/mL).
Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP (Cat. #AP124P) and a chemiluminescence detection system (Please see figures).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Chromatin Biology
This ChIPAb+ Validated Antibody & Primer Set conveniently includes the antibody, matched IgG negative control antibody & set control PCR primers that detect a known positive locus.
Biochem/physiol Actions
Recognizes histone H3, Mr 17 kDa, monomethylated at Lys9.
The immunogen sequence is identical in a wide range of animal and plant species.
Packaging
25 assays per kit, ~2μg per chromatin immunoprecipitation
Physical form
Anti-monomethyl-Histone H3 (Lys9) (mouse monoclonal IgG2aқ, clone CMA306). One vial containing 50 μg of protein G purified antibody in 50 μL PBS containing 0.05% sodium azide. Store at -20°C.
Normal Mouse IgG. Two vials containing 25 μg purified Mouse IgG in 25 μL storage buffer containing 0.1% sodium azide. Store at -20°C.
ChIP Primers GAPDH Coding region. One vial containing 75 μL of 5 μM of each primer specific for a region of the human GAPDH coding region. Store at -20°C.
FOR: GGC TCC CAC CTT TCT CAT CC
REV: GGC CAT CCA CAG TCT TCT GG
Preparation Note
Stable for 1 year at -20°C from date of receipt. Aliquot upon thawing, avoid freeze thaw cycles.
Analysis Note
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 106 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either a normal mouse IgG or Anti-monomethyl-Histone H3 (Lys9) antibody and the Magna ChIP G (Cat. #17-611) Kit. Successful immunoprecipitation of monomethyl-histone H3 (Lys9) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH Coding region (Please see figures).
Please refer to the EZ-Magna G ChIP (Cat. #17-409) or EZ-ChIP (Cat. #17-371) protocol for experimental details.
Control
Included negative control mouse IgG antibody and control primers specific for human GAPDH coding region.
Legal Information
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.
