ChIPAb+ Ubiquityl-Histone H2B - ChIP Validated Antibody and Primer Set

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+ Ubiquityl-Histone H2B, (clone 56) set includes the Ubiquityl-Histone H2B, (clone 56) antibody, a negative control antibody (purified Mouse IgG), and qPCR primers which amplify a 213 bp region within the coding region of the human GAPDH gene.
The ubiquityl-Histone H2B and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Ubiquityl-Histone H2B associated chromatin.

Specificity

Not tested in other species. The immunogen sequence is identical in a wide range of animal and plant species, so broad cross-reactivity is expected.

Recognizes histone H2B ubiquitinated on lysine 120 but not free ubiquitin or histone H2B in its unmodified state.

Immunogen

Epitope: Lys120 ubiquitination site

Immunogen used was a branched peptide corresponding to the ubiquitination site on human histone H2B.

Application

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa S3 cells (1 X 10⁶ cell equivalents per IP) was subjected to chromatin immunoprecipitation using 1 μg of either a normal mouse IgG or Anti-Ubiquityl-Histone H2B antibody and the Magna ChIP G Kit (Cat. # 17-611).
Successful immunoprecipitation of ubiquityl-Histone H2B associated DNA fragments was verified by qPCR using GAPDH promoter (negative) and GAPDH coding (positive) 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. # 17-409) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.

Western Blot Analysis:
HeLa cell lysate was resolved by electrophoresis, transferred to PVDF and probed with anti-Ubiquityl-histone H2B at a dilution of 1:4000 (Please see figures).

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Chromatin Biology

Histones

This ChIPAb+ Ubiquityl-Histone H2B -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Packaging

25 assays per set. Recommended use: ~1 μg antibody per chromatin immunoprecipitation (dependent upon biological context).

Quality

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa S3 cells (1X 10⁶ cell equivalents per IP) were subjected to chromatin immuno-precipitation using 1 µg of either a normal mouse IgG or Anti-ubiquityl-Histone H2B, clone 56 antibody and the Magna ChIP^® G Kit (Cat. # 17-611). Successful immunoprecipitation of ubiquityl-Histone H2B-associated DNA fragments was verified by qPCR using Control Primers.
Please refer to the EZ-Magna ChIP G (Cat. # 17-409) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.

Target description

~25 kDa

Physical form

Anti-ubiquityl-Histone H2B, clone 56 (mouse monoclonal IgG). One vial containing 25 μg of protein G purified antibody in 25 μg in 50 μL PBS containing 0.05% sodium azide. Store at -20°C.

Normal Mouse IgG. One vial 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 the coding region of human GAPDH. Store at -20°C.
FOR: GGC TCC CAC CTT TCT CAT CC
REV: GGC CAT CCA CAG TCT TCT GG

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 mouse IgG antibody and primers specific for human GAPDH coding region.

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.