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 H3 (Lys36) set includes the monomethyl-Histone H3 (Lys36) antibody, a Normal Rabbit IgG, and control primers which amplify a 213 bp region of the human GAPDH coding region. The Monomethyl-Histone H3 (Lys36) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Monomethyl-Histone H3 (Lys36) associated chromatin.
~17 kDa
Immunogen
Epitope: Monomethylated Histone H3 Lys36
KLH-conjugated linear peptide corresponding to the N-terminus of human Histone H3 monomethylated at Lys36.
Application
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µg of Normal Rabbit IgG, or 3 µL (1 µg) Anti-Monomethyl-Histone H3 (Lys36) and the Magna ChIP HiSens (Cat. # 17-10460). Successful immunoprecipitation of monomethyl-Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 as a positive locus, and GAPDH promoter as a negative locus.
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 Magna ChIP HiSens (Cat. # 17-10460) or EZ-MagnaChIP HiSens(Cat. # 17-10461) protocol for experimental details.
Western Blotting Analysis:
HeLa acid extract and various recombinant Histone proteins were probed with Anti-monomethyl-Histone H3 (Lys36) (0.05 µg /mL).
Immunoprecipitation Analysis:
10 µg from a representative lot immunoprecipitated monomethyl-Histone H3 (Lys36) from HeLa acid extract.
10 µg of this antibody immunoprecipitated monomethyl -Histone H3 (Lys36) from 500 µg of HeLa acid extract.
Arrow indicates monomethyl-Histone H3 (Lys36) (~17 kDa).
Dot Blot Specificity Analysis:
Unmodified and various modified Histone peptides were probed with Anti-monomethyl-Histone H3 (Lys36) (0.5 µg/mL).Histone peptides with various modifications were transferred to PVDF membrane (Cat. #: 16-665) and probed with Anti-monomethyl-Histone H3 (Lys36) at (0.5 µg/mL). 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
Histones
Chromatin Biology
The ChIPAb+ Monomethyl-Histone H3 (Lys36) set includes the monomethyl-Histone H3 (Lys36) antibody, a Normal Rabbit IgG & control primers which amplify a 213 bp region of the human GAPDH coding region.
Biochem/physiol Actions
This antibody recognizes the N-terminus of Histone H3 monomethylated at Lys36.
Wide species cross reactivity is predicted based on sequence similarity.
Packaging
25 assays per set. Recommended use: 3 μL (1 μg) of antibody per chromatin immunoprecipitation (dependent upon biological context).
Physical form
Anti-Monomethyl-Histone H3 (Lys36) (Rabbit Polyclonal). One vial containing 75 µL 0.35 mg/mL purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH7.4),150mM NaCl, and 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C.
Normal Rabbit IgG. One vial containing 125 µg Rabbit IgG in 125 µL storage buffer containing 0.05% sodium azide. Store at -20°C.
ChIP Primers, GAPDH coding D2. One vial containing 75 μL of 5 μM of each primer specific for human GAPDH coding region. Store at -20°C.
FOR: GCC ATG TAG ACC CCT TGA AGA G
REV: ACT GGT TGA GCA CAG GGT ACT TTA T
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 (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µg of either Normal Rabbit IgG, or 3 µL (1 µg) Anti-Monomethyl-Histone H3 (Lys36) and the Magna ChIP® HiSens Kit (Cat. # 17-10460).
Successful immunoprecipitation of monomethyl Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2. Please refer to the MagnaChIP HiSens (Cat. # 17-10460) or EZ-MagnaChIP HiSens (Cat. # 17-10461) protocol for experimental details.
Control
Includes normal rabbit IgG 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.
