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+ Monomethyl-Histone H4 (Lys20) set includes the Anti-monomethyl-Histone H4 (Lys20) antibody, a negative control antibody (purified Rabbit IgG), and qPCR primers, which amplify a 212 base pair PCR product within the human GAPDH coding region. The monomethyl histone H4 (Lys20) and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of monomethyl-histone H4 (Lys20) associated chromatin.
~11 kDa
Immunogen
Epitope: a.a. 15-24
The immunogen was a synthetic peptide corresponding to amino acids 15-24 of histone H4, monomethylated on Lys20.
Application
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (2 X 10⁶ cell equivalents per IP) was subjected to chromatin immunoprecipitation using 4 μg of either a negative control antibody or Anti-Monomethyl-Histone H4
(Lys20) antibody and the Magna ChIP A Kit (Cat. #17-610). Successful immunoprecipitation of monomethyl-histone H4 (Lys20)-associated DNA fragments was verified by qPCR using GAPDH coding region ChIP Primers versus Control Primers corresponding to the GAPDH promoter (Please see figures). Data is presented as percent input of each IP sample relative to input chromatin, with immunoprecipitated DNA from negative control antibody shown as (-) and monomethyl-histone H4 (Lys20) shown as (+).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Western Blot Analysis:
0.1 μg/mL of proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system (Please see figures)
Peptide Blocking Assay:
40 μg of histone H4 peptide containing monomethyl Lys20 abolished detection of histone H4 by anti-monomethyl-Histone H4 (Lys20) in immunoblot analysis of acid extracts of HeLa cells (Please see figures).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Chromatin Biology
Histones
This ChIPAb+ Monomethyl-Histone H4 (Lys20) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Biochem/physiol Actions
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 H4, Mr ~11 kDa.
Packaging
25 assays per set. Recommended use: ~4 μg antibody per chromatin immunoprecipitation (dependent upon biological context).
Physical form
Anti-monomethyl-Histone H4 (Lys20) (rabbit polyclonal IgG). One vial containing 100 μg of affinity purified antibody in 100 μL 0.1M Tris-Glycine (pH 7.4), 15 mM NaCl, and 0.05% NaN3. Store at -20°C.
Normal Rabbit IgG. One vial containing 125 μg of purified rabbit IgG in 125 μL storage buffer. Store at -20°C.
ChIP Primers GAPDH Coding Region. One vial containing 75 μL of 5 μM of each primer specific for human GAPDH coding region.Store at -20°C.
FOR: GGC TCC CAC CTT TCT CAT CC
REV: GGC CAT CCA CAG TCT TCT GG
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.
Analysis Note
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 μg of either a negative control antibody or Anti-monomethyl-Histone H4 (Lys20) antibody and the Magna ChIP A Kit (Cat. # 17-610). Successful immuno-precipitation of monomethyl-histone H4 (Lys20)-associated DNA fragments was verified by qPCR using ChIP Primers GAPDH Coding region (Please see figures).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Control
Includes negative control rabbit IgG antibody and control primers specific for human GAPDH coding region.
Legal Information
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.
