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Analysis Note

ControlIncludes negative control mouse IgG antibody and control primers specific for human β-globin promoter.

Application

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

Research CategoryEpigenetics & Nuclear Function

Research Sub CategoryChromatin Biology

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (2 X 10⁶ cell equivalents per IP) was subjected to chromatin immunoprecipitation using 2 µg of either normal mouse IgG or Anti- Histone H3 (Unmodified Lys4) antibody and the Magna ChIP G Kit (Cat. #17-611). Successful immunoprecipitation of histone H3 associated DNA fragments was verified by qPCR using β-globin ChIP Primers versus GAPDH Promoter Control Primers. Data is presented as percent input of each IP sample relative to input chromatin, with the indicated primer sets (Please see figures).

Western Blot Analysis:
Acid extracted proteins from HeLa cells (Lane 1) and Recombinant Histones (Lane 2-6) were resolved by electrophoresis, transferred to PVDF membrane and probed with anti-Histone H3 (Unmodified Lys4) (0.1 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).

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+ Histone H3 (Unmodified Lys4) set includes the Anti-Histone H3 (unmodified Lys4) antibody, a negative control antibody (purified Mouse IgG), and qPCR primers which amplify a 110 bp region within the promoter of the human β-globin gene. The histone H3 (Unmodified Lys4) and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of histone H3 (Unmodified Lys4)-associated chromatin.

Immunogen

Epitope: a.a. 1-12

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

Legal Information

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

Packaging

25 assays per kit, ~2µg per chromatin immunoprecipitation

Physical form

Anti-Histone H3 (Unmodified Lys4) (mouse monoclonal IgG2bκ, clone CMA301). 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 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

Quality

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-Histone H3 (Unmodified Lys4) antibody and the Magna ChIP G Kit (Cat. #17-611). Successful immunoprecipitation of histone H3 (Unmodified Lys4) associated DNA fragments was verified by qPCR using β-Globin ChIP 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

The peptide sequence is identical in a wide range of animal and plant species, so broad cross-reactivity is expected.

Recognizes unmodified histone H3 (Lys4), Mr 17 kDa. Epitope specificity of antibody as determined by ELISA maps near Lys4. Antibody tolerates modifications on Lys9 and Ser10 but not Lys4 or Thr31.

Storage and Stability

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

Target description

Histone H3 at ~17 kDa