Analysis Note

ControlHeLa cell acid extracts

Application

Research Sub CategoryHistones

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. 04-808, Anti-dimethyl H3 (Arg2) at 1ug/mL (1:1000) dilution. Proteins were visualized using a Donkey anti-rabbit IgG conjugated to HRP and a chemiluminescence detection system.

Peptide Inhibition Analysis:
2 µM of histone H3 peptide containing dimethyl-Arg2 abolished detection of histone H3 by a previous lot of anti-dimethyl-Histone H3(Arg2) (1:4,000 dilution) in immunoblots of HeLa acid extracts. Some competition of signal was also seen with unmodified peptide containing Arg2.

Peptide Dot Blot Analysis:
A 1:4,000-1:16,000 dilution of a previous lot only detected histone H3 peptide containing dimethyl-Arg2. Peptides containing unmodi-fied Arg2, dimethyl-Arg17 or dimethyl-Arg26 were not detected.

Beadlyte™ Histone Peptide Assay:
1:500-1:48,000 dilutions of a previous lot detected histone H3 peptide containing dimethyl-Arg2. Cross-reactivity to peptides containing unmodified Arg2 or dimethylArg 17 or 26 was not seen.

Use Anti-dimethyl-Histone H3 (Arg2), clone 20.2 Antibody (rabbit monoclonal antibody) validated in DB, Mplex, PIA, WB to detect dimethyl-Histone H3 (Arg2) also known as H3R2me2, Histone H3 (di methyl R2).

Research CategoryEpigenetics & Nuclear Function

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

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. 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. Histone H3 Arg2 methylation is a widespread silencing modification that inhibits histone H3 Lys4 trimethylation.

Immunogen

KLH-conjugated, synthetic peptide containing the sequence R30;Ame2RTKQR30;, in which me2R corresponds to dimethyl-arginine 2 of human histone H3.

Epitope: Arg2 of Histone H3

Legal Information

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

Linkage

Replaces: 05-808

Physical form

Cultured supernantant in 0.05% sodium azide

Quality

Routinely evaluated by immunoblot on acid-extracted proteins from HeLa cells.

Immunoblot Analysis: A 1:500 (lane 1) and 1:1000 (lane 2) dilution of this lot detected methylated Histone H3 in acid-extracted proteins from HeLa cells.

Specificity

Recognizes dimethyl-arginine at residue 2 of histone H3, Mr 17kDa. Additional unidentified bands above 50 kDa are detected in some samples.

The immunizing sequence is highly conserved evolutionarily, so broad cross-reactivity is expected.

Storage and Stability

Stable for 1 year at -20°C from date of receipt.
Handling Recommendations: Upon receipt, 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

17kDa