Analysis Note

ControlIncludes normal rabbit serum and primers specific for human GAPDH, coding region.

Application

Research Sub CategoryHistones

Research CategoryEpigenetics & Nuclear Function

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP™ HiSens (Cat. # 17-10460). Successful immunoprecipitation of dimethyl Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323) as a positive locus, and GAPDH promoter (Part No. 22-004) as a negative locus. (Figure 2). Data are 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.

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP™ HiSens (Cat. # 17-10460). Successful immunoprecipitation of dimethyl Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323) as a positive locus, and GAPDH promoter (Part No. 22-004) as a negative locus. Please refer to the Magna ChIP™ HiSens (Cat. # 17-10460) or EZ-MagnaChIP™ HiSens(Cat. # 17-10461) protocol for experimental details.

Western Blotting Analysis:
A 1:10,000 dilution of this antibody detected dimethyl Histone H3 (Lys36) in 10 µg HeLa acid extract and demonstrated a loss of signal in 0.5 µg of unmethylated recombinant Histone H3

Dot Blot Analysis: Representative lot data.
Absurance™ Histone H3 Antibody Specificity Array (Cat. No. 16-667, Fig. A) Absurance™ Histone H2A, H2B, and H4 Antibody Specificity Array (Cat. No. 16-665, Fig. B) were probed with Anti-Dimethyl Histone H3 (Lys36) (1:10,000 dilution). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system.

This ChIPAb+ Validated Antibody & Primer Set conveniently includes the antibody, matched IgG negative control antibody & set control PCR primers that detect a known positive locus.

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+™ Dimethyl Histone H3 (Lys36) set includes a Dimethyl Histone H3 (Lys36) antibody, a Normal Rabbit Serum, and positive control primers which amplify a 87 bp region of the human GAPDH coding region. The Dimethyl Histone H3 (Lys36) antibody and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of dimethyl Histone H3 (Lys36) associated chromatin.

Histones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin. The four core histones, H2A, H2B, H3, and H4, assemble into an octamer (2 molecules of each). Subsequently, 146 base pairs of DNA are wrapped around the octamer, forming a nucleosome, the basic subunit of chromatin. Histones are modified post-translationally by the actions of enzymes in both the nucleus and cytoplasm. These modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. These modifications can alter local chromatin architecture, or recruit trans-acting factors that recognize specific histone modifications (the "histone code" hypothesis). The modifications occur predominantly on the N-terminal and C-terminal tails that extend beyond the nucleosome core particle. Methylation of histone H3 on Lys36 (H3K36me2/3) is tightly associated with actively transcribed genes, and this modification is found primarily within the coding region, suggesting H3K36 methylation is necessary for efficient RNA polymerase II elongation and processivity.

Immunogen

KLH-conjugated linear peptide corresponding to a region near the N-terminus of human Histone H3 dimethylated at Lys36.

Epitope: Dimethylated 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

Recommended use: 1 µL of antibody per chromatin immunoprecipitation (dependent upon biological context).

Physical form

Anti-Dimethyl Histone H3 (Lys36) (Rabbit Polyclonal), Part No. CS207357. One vial containing 25 µL rabbit polyclonal serum containing 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C.

Normal Rabbit Serum, Part No. CS200585. One vial containing 25 µL antiserum containing 0.05% sodium azide. Store at -20°C.

ChIP Primers, GAPDH coding D2. Part No. CS207323. One vial containing 75 µL of 5 µM of each primer specific for human GAPDH coding region. (chr12:6647453+6647539, hg 19 build) Store at -20°C.
FOR: 5' GCC ATG TAG ACC CCT TGA AGA GC 3'
REV: 5' ACT GGT TGA GCA CAG GGT ACT TTA T 3'

Quality

Sonicated chromatin prepared from HeLa cells (1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 1 µL of either Normal Rabbit Serum (Part No. CS200585), or 1 µL Anti-Dimethyl Histone H3 (Lys36) (Part No.CS207357) and the Magna ChIP^® HiSens Kit (Cat. # 17-10460). Successful immunoprecipitation of dimethyl-Histone H3 (Lys36) associated DNA fragments was verified by qPCR using ChIP Primers, GAPDH coding D2 (Part No. CS207323).
Please refer to the Magna ChIP™ HiSens (Cat. # 17-10460) or EZ-Magna ChIP™ HiSens (Cat. # 17-10461) protocol for experimental details.

Specificity

Recognizes Dimethyl Histone H3 (Lys36). No detectable cross-reactivity unmethylated, mono, or dimethylated Lys36. No detectable cross reactivity to other histone modifications as measured by AbSurance Histone Antibody Specificity Arrays.

Wide reactivity with other species is expected.

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