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Analysis Note

ControlIncluded negative control mouse IgG antibody and control primers specific for human GAPDH promoter.

Application

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from untreated or colcemid treated 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-trimethyl-Histone H3 (Lys4) antibody and the Magna ChIP G Kit (Cat. #17-611). Successful immunoprecipitation of trimethyl-Histone H3 (Lys4) associated DNA fragments was verified by qPCR using primers ampliflying a region of the human B-Globin promoter or using GAPDH promoter Control Primers (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 lot data.
HeLa acid extract were resolved by electrophoresis, transferred to PVDF membrane and probed with Anti-trimethyl Histone H3 (Lys4), clone CMA304 at 1 µg/ml (lane 1) or 0.5 µg/ml (lane 2). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP (Cat. #AP124P) and a chemiluminescence detection system (Please see figures).

Research CategoryEpigenetics & Nuclear Function

Research Sub CategoryChromatin Biology

This ChIPAb+ Trimethyl-Histone H3 (Lys4) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

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

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 (Lys4) set includes the Anti-trimethyl-Histone H3 (Lys4) antibody, a negative control antibody (purified Mouse IgG), and qPCR primers which amplify a 166 bp region within the promoter of the human GAPDH gene. The trimethyl-Histone H3 (Lys4) 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 (Lys4)-associated chromatin.

Immunogen

The trimethyl-histone H3 (Lys4) purified antibody is made against a synthetic peptide (trimethylated at Lys4) corresponding to amino acids 1-12 of human histone H3.

Epitope: a.a. 1-12

Legal Information

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

Packaging

25 assays per kit, ~2µg per chromatin immunoprecipitation

Physical form

Anti-trimethyl-Histone H3 (Lys4) (mouse monoclonal IgG1, Clone CMA304). One vial containing 50 µg of protein G purified antibody in 50 µL PBS containing 0.05% sodium. 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.

Control Primers. One vial containing 75 µL of 5 µM of each primer specific for the promoter region of GAPDH. Store at -20°C.
FOR: TAC TAG CGG TTT TAC GGG CG
REV: TCG AAC AGG AGG AGC AGA GAG CGA

Quality

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from colcemid-reated HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either normal mouse IgG or Anti-trimethyl-Histone H3 (Lys4) antibody and the Magna ChIP G Kit (Cat. #17-611). Successful immuno-precipitation of trimethyl-histone H3 (Lys4) associated DNA fragments was verified by qPCR using Control Primers (Please see figures).
Please refer to the EZ-Magna G ChIP™ (Cat. #17-409) or EZ-ChIP™ (Cat. #17-371) protocol for experimental details.

Specificity

Recognizes histone H3, Mr 17 kDa, trimethylated at Lys4.

The immunogen sequence is identical in a wide range of animal and plant species.

Storage and Stability

Stable for 1 year at -20°C from date of receipt. Aliquot upon thawing, avoid freeze thaw cycles.

Target description

Trimethyl histone H3 at ~17 kDa