Analysis Note

ControlUV-treated 293 cell extracts, UV-treated HeLa cell extracts, breast cancer tissue, HEPG2 cell extracts.

Application

Flow Cytometry:
This antibody has been reported by an independent laboratory to detect phosphorylated histone H3 using flow cytometry.

Beadlyte™ Histone-Peptide Specificity Assay:
1:1,000 to 1:81,000 dilutions of a previous lot were incubated with histone H3 peptides containing various modifications conjugated to Luminex™ microspheres. (Figure B). Only the peptide containing phospho-serine 10 was detected.


Immunocytochemistry:
0.2 µg/mL of a previous lot showed positive chromosome immunostaining for mitotic A431 and HeLa cells fixed with 95% ethanol and 5% acetic acid and permeabilized with 0.1% Triton™ X-100.

Peptide Inhibition Analysis:
Detection of histone H3 in immunoblots of colcemid treated HeLa acid extracts by anti-phospho-Histone H3 (Ser10) was diminished by 10 µM of histone H3 peptide containing phospho-serine 10, but not by peptides containing phospho-serine 28 or an unmodified histone H3 sequence (Figure C).

Immunofluorescence:

Anti-phospho-Histone H3 (Ser10) Antibody, clone 3H10 is a mouse monoclonal antibody for detection of Histone H3 phosphorylated at serine 10. This purified mAb, also known as H3S10p, is published in peer reviewed journals and has been validated in FC, ICC, IF, PIA, WB, Mplex.

General description

Histone H3 is one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N-terminal tail, H3 is involved with the structure of the nucleosomes of the ′beads on a string′ structure. The N-terminal tail of histone H3 protrudes from the globular nucleosome core and can undergo several different types of epigenetic modifications that influence cellular processes. These modifications include the covalent attachment of methyl or acetyl groups to lysine and arginine amino acids and the phosphorylation of serine or threonine.

Legal Information

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

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Purified mouse IgG1k in buffer containing 0.1 M Tris-glycine, pH 7.4, 0.15 M NaCl and 0.05% sodium azide.

Format: Purified

Quality

Routinely evaluated by western blot acid extracted proteins from mitotic HeLa cells treated with colcemid (Catalog # 17-306).

Western Blot Analysis:
0.05-0.5 µg/mL of this lot detected phosphorylated histone H3 in acid extracted proteins from mitotic HeLa cells treated with colcemid (Catalog # 17-306), but not unmodified recombinant Histone H3 (Catalog # 14-494) (Figure A).

Specificity

Broad species cross-reactivity is expected.

Histone H3 phosphorylated at Ser10.

Storage and Stability

Stable for 1 year at 2 to 8°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. Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.

Target description

17 kDa