Analysis Note

ControlIncludes normal rabbit IgG and primers specific for human GAPDH coding region

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.

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.

Research CategoryEpigenetics & Nuclear Function

Research Sub CategoryHistonesChromatin Biology

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.

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.

Immunogen

KLH-conjugated linear peptide corresponding to the N-terminus of human Histone H3 monomethylated at Lys36.

Epitope: Monomethylated Histone H3 Lys36

Legal Information

MAGNA CHIP is a registered trademark of Merck KGaA, Darmstadt, Germany

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

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

Quality

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.

Specificity

This antibody recognizes the N-terminus of Histone H3 monomethylated at Lys36.

Wide species cross reactivity is predicted based on sequence similarity.

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.

Target description

~17 kDa