ChIPAb+ Histone H3 (CT) - ChIP Validated Antibody and Primer Set, rabbit monoclonal

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 (C-term) set includes the Histone H3 (C-term) antibody, a negative control supernatant (rabbit), and qPCR primers which amplify a 110 bp region of human β-Globin. The Histone H3 (C-term) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Histone H3-associated chromatin.

Histone H3 is one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N-terminal tail, H3 is involved with the structure of the nucleosomes of the ′beads on a string′ structure. Histone proteins are highly post-translationally modified, and Histone H3 is the most extensively modified of the five histones. The term "Histone H3" alone is purposely ambiguous in that it does not distinguish between sequence variants or modification state. Histone H3 is an important protein in the emerging field of epigenetics, where its sequence variants and variable modification states are thought to play a role in the dynamic and long term regulation of genes.

Specificity

Broad species cross-reactivity expected due to sequence homology.

Recognizes Histone H3 near the C-terminus.

Immunogen

Epitope: C-Terminus

KLH-conjugated, synthetic peptide corres-ponding to the C-terminus of human Histone H3.

Application

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 µL of a negative control supernatant, or 4 µL of Anti-Histone H3 (C-term) and the Magna ChIP^® A Kit (Cat. # 17-610).
Successful immunoprecipitation of Histone H3 associated DNA fragments was verified by qPCR using ChIP Primers, β-Globin as well as GAPDH promoter 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 ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.

Western Blot Analysis:
Representative Lot Data.
Acid extracts from colcemid treated HeLa cells were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-Histone H3 (C-term) (1:1000 dilution).
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system. (Please see figures).


Western Blot Analysis:
Representative lot data.
Acid extracted proteins from HeLa cells untreated (Lane 1), colcemid treated (Lane 2). HeLa nuclear extracts (Lane 3), acid extracted HeLa cells treated with sodium butyrate (Lane 4), untreated (Lane 5) and unmodified recombinant Histone H3 (Lane 6) were resolved by electrophoresis, transferred to nitrocellulose and probed with a previous lot of anti-Histone H3 (C-term) (1:5000 dilution).

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
Histones

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

Packaging

25 assays per set. Recommended use: ~4 μL of antibody per chromatin immunoprecipitation (dependent upon biological context).

Quality

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 µL of either Negative Control Supernatant or 4 µL of Anti-Histone H3 (C-term) and the Magna ChIP^® A Kit (Cat. # 17-610). Successful immunoprecipitation of Histone H3 associated DNA fragments was verified by qPCR using ChIP Primers, 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.

Target description

~17 kda

Physical form

Anti-Histone H3 (C-term) (rabbit monoclonal IgG). One vial containing 100 µL of cultured supernatant in 0.05% sodium azide. Store at -20°C.

Negative Control Supernatant (rabbit). One vial containing 100 µL of cultured supernatant in 0.05% sodium azide. Store at -20°C.

ChIP Primers, β-Globin.
One vial containing 75 μL of 5 μM of each primer specific for human β-Globin. Store at -20°C.
FOR: AGG ACA GGT ACG GCT GTC ATC
REV: TTT ATG CCC AGC CCT GGC TC

Storage and Stability

Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, 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

Control
Includes negative control supernatant (rabbit) and primers specific for human β-Globin.

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.