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+ Trimethyl-Histone H4 (Lys20) set includes the Anti-trimethyl-Histone H4 (Lys20) antibody, a negative control supernatant, and qPCR primers which amplify a 110 bp region within the promoter of the humanβ-globin gene. The trimethyl 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 trimethyl-histone H4 (Lys20) associated chromatin.
~11 kDa
Immunogen
Epitope: a.a. 18-22
The trimethyl histone H4 (Lys20) antibody is made against a synthetic peptide corresponding to amino acids 18-22 of histone H4. trimethylated on lysine 20.
Application
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (2 X 10⁶ cell equivalents per IP) was subjected to chromatin immunoprecipitation using 10 μL of either a negative control supernatant or Anti-Trimethyl-Histone H4 (Lys20) antibody and the Magna ChIP A Kit (Part No. 17-610). Successful immunoprecipitation of trimethylhistone H4 (Lys20) associated DNA fragments was verified by qPCR using β-globin 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 supernatant shown as (-) and trimethyl 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:
Acid extracts from HeLa cells untreated or pre-absorbed with 1 μM of trimethylated (Lys20) Histone H4 peptide (Lanes 1 and 2 respectively) were probed with anti-trimethyl-Histone H4 (Lys20) (1:1,000) (Please see figures).
Dot Blot Analysis: Absurance Histone H3 Antibody Specificity Array (Cat. No. 16-667) and Absurance Histone H2A, H2B, H4 Antibody Specificity Array (Cat. No. 16-665), which contain histone peptides with various modifications were probed with Cat. No 04-079, Anti-trimethyl-Histone H4 (Lys20) ; dilution 1:500. Proteins were visualized using a Donkey anti-rabbit IgG conjugated to HRP and a chemiluminescence detection system.
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Chromatin Biology
Histones
This ChIPAb+ Trimethyl-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
10 assays per set. Recommended use: ~10 μL antibody per chromatin immunoprecipitation (dependent upon biological context).
Physical form
Anti-trimethyl-Histone H4 (Lys20) (rabbit monoclonal IgG). One vial containing 100 μL of cell culture supernatant in 0.1% sodium azide. Store at -20°C.
Negative Control Supernatant. One vial containing 100 μL cell rabbit monoclonal culture supernatant containing 0.1% sodium azide. Store at -20°C.
ChIP Primers B-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
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 10 µL of either a negative control supernatant, or Anti-Trimethyl-Histone H4 (Lys20) antibody and the Magna ChIP® A Kit (Cat. # 17-610).
Successful immunoprecipitation of trimethyl-histone H4 (Lys20)-associated DNA fragments was verified by qPCR using ChIP Primers B-Globin.
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 antibody and control primers specific for human β-globin promoter.
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.
