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+ HDAC2 set includes the HDAC2 antibody, a Normal Mouse IgG, and control primers which amplify a 92 bp region of ChIP Primers, VWF promoter. The HDAC2 and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of HDAC2 -associated chromatin.
Histone deacetylase 2 (HDAC2), or transcriptional regulator homolog RPD3 L1, is highly homologous to the yeast transcription factor RPD3 (reduced potassium dependency 3) gene. As in yeast, human HDA2 is likely to be involved in regulating chromatin structure during transcription. It has been implicated to associate with YY1, a mammalian zinc-finger transcription factor, which negatively regulates transcription by tethering RPD3 to DNA as a cofactor. This process is highly conserved from yeast to human.
~60 kDa
Immunogen
Epitope: C-terminus
KLH-conjugated, synthetic peptide (CEKTDTKGTKSEQLSNP) corresponding to human HDAC2 at the C-terminus.
Application
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from untreated or UV treated (6 hrs, 50 joules/m2.) U2OS cells (3 X 10E6 cell equivalents per IP) was subjected to chromatin immunoprecipitation using using 2 µg of either Normal Mouse IgG or 2 µg of Anti-HDAC2 and the Magna ChIP A/G Kit (Cat. # 17-10085). Successful immunoprecipitation of HDAC2 associated DNA fragments was verified by qPCR using ChIP Primers, VWF promoter. Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated. (Figure 2).
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.
HeLa nuclear extract was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-HDAC2 (0.5 μg/mL). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates HDAC2 (~60 kDa). (Figure 3).
Immunoprecipitation and HDAC Assay:
Representative lot data.
3 μL of a previous lot was used to immunoprecipitate HDAC activity from HeLa nuclear extract, which was then measured using the HDAC Assay Kit (Fluorometric Detection) (Catalog # 17-356) (Figure 4).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Histones
This ChIPAb+ HDAC2 -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Biochem/physiol Actions
Predicted to cross-react with rat based on sequence homology.
This antibody recognizes HDAC2 at the C-terminus.
Packaging
25 assays per set. Recommended use: ~2 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).
Physical form
Anti-HDAC2 (mouse monoclonal). One vial containing 50 µg of purified mouse monoclonal IgG in buffer containing 70% storage buffer (0.1M Tris-glycine, pH 7.4, 0.15 M NaCl, 0.05% sodium azide) and 30% glycerol. Store at -20° C.
Concentration: 0.94 mg/mL
Normal Mouse IgG. One vial containing 125 µg of purified mouse IgG in 125 µL of storage buffer containing 0.1% sodium azide. Store at -20°C.
ChIP Primers, VWF promoter. One vial containing 75 μL of 5 μM of each primer specific for the promoter region of human von Willebrand factor. Store at -20°C.
FOR: GCT GAG AGC ATG GCC TAG GGT GGT GGG CGG CAC
REV: CCC CTG CAA ATG AGG GCT GCG GCT ATC TCC AAG
Format: Purified
Protein G Purified
Preparation Note
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.
Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.
Analysis Note
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HEK293T cells (5 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either Normal Mouse IgG, or 2 µg of Anti-HDAC2 and the Magna ChIP® A/G Kit (Cat. # 17-10085). Successful immunoprecipitation of HDAC2 associated DNA fragments was verified by qPCR using ChIP Primers, VWF promoter (Figure 1).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details
Control
Includes normal mouse IgG and primers specific for human von Willebrand factor.
Other Notes
Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
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.
