H2AFX antibody (pSer139)

Catalog No. ABIN967669

Product Details anti-H2AFX Antibody

Target: H2AFX (H2A Histone Family, Member X (H2AFX))

Binding Specificity: pSer139

Reactivity: Human, Mouse

Host: Mouse

Clonality: Monoclonal

Conjugate: This H2AFX antibody is un-conjugated

Application: Western Blotting (WB), BioImaging (BI)

Brand: BD Pharmingen™

Cross-Reactivity: Mouse (Murine)

Characteristics: 1. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
2. Triton is a trademark of the Dow Chemical Company.
3. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
4. Please refer to us for technical protocols.

Purification: The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Immunogen: Phosphorylated Human H2AX Peptide

Clone: N1-431

Isotype: IgG1 kappa

Application Details

Application Notes: Recommended Assay Procedure for Bioimaging:
1. Seed the cells in appropriate culture medium at an appropriate cell density in an 96-well Imaging Plate , and culture overnight to 48 hours.
2. Remove the culture medium from the wells, and wash (one to two times) with 100 myl of 1× PBS.
3. Fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or fixation buffer to each well and incubating for 10 minutes at room temperature (RT).
4. Remove the fixative from the wells, and wash the wells (one to two times) with 100 myl of 1× PBS.
5. Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c): a. Add 100 µl of -20°C 90% methanol to each well and incubate for 5 minutes at RT. b. Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT. c. Add 100 µl of 1× Perm/Wash buffer to each well and incubate for 15 to 30 minutes at RT. Continue to use 1× Perm/Wash buffer for all subsequent wash and dilutions steps.
6. Remove the permeabilization buffer from the wells, and wash one to two times with 100 myl of appropriate buffer (either 1× PBS or 1× Perm/Wash buffer, see step 5.c.).
7. Optional blocking step: Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.
8. Dilute the antibody to its optimal working concentration in appropriate dilution buffer. Titrate purified (unconjugated) antibodies and second-step reagents to determine the optimal concentration. If using a Bioimaging Certified antibody conjugate, dilute it 1:10.
9. Add 50 µl of diluted antibody per well and incubate for 60 minutes at RT. Incubate in the dark if using fluorescently labeled antibodies.
10. Remove the antibody, and wash the wells three times with 100 myl of wash buffer. An optional detergent wash (100 myl of 0.05% Tween in 1× PBS) can be included prior to the regular wash steps.
11. If the antibody being used is fluorescently labeled, then move to step 12. Otherwise, if using a purified unlabeled antibody, repeat steps 8 to 10 with a fluorescently labeled second-step reagent to detect the purified antibody.
12. After the final wash, counter-stain the nuclei by adding 100 ml of a 2 mg/ml solution of Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
13. View and analyze the cells on an appropriate imaging instrument.

Comment:

Related Products: ABIN967389

Restrictions: For Research Use only

Handling

Format: Liquid

Concentration: 0.5 mg/mL

Buffer: Aqueous buffered solution containing ≤0.09 % sodium azide.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C

Storage Comment: Store undiluted at 4°C.

References for anti-H2AFX antibody (ABIN967669)

Kuo, Yang: "Gamma-H2AX - a novel biomarker for DNA double-strand breaks." in: In vivo (Athens, Greece), Vol. 22, Issue 3, pp. 305-9, (2008) (PubMed).

Fernandez-Capetillo, Lee, Nussenzweig, Nussenzweig: "H2AX: the histone guardian of the genome." in: DNA repair, Vol. 3, Issue 8-9, pp. 959-67, (2004) (PubMed).

Burma, Chen, Murphy, Kurimasa, Chen: "ATM phosphorylates histone H2AX in response to DNA double-strand breaks." in: The Journal of biological chemistry, Vol. 276, Issue 45, pp. 42462-7, (2001) (PubMed).

Rogakou, Nieves-Neira, Boon, Pommier, Bonner: "Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139." in: The Journal of biological chemistry, Vol. 275, Issue 13, pp. 9390-5, (2000) (PubMed).

Rogakou, Pilch, Orr, Ivanova, Bonner: "DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139." in: The Journal of biological chemistry, Vol. 273, Issue 10, pp. 5858-68, (1998) (PubMed).

Target Details for H2AFX

Target: H2AFX (H2A Histone Family, Member X (H2AFX))

Alternative Name: H2AX (H2AFX Products)

Synonyms: H2A.X antibody, H2A/X antibody, H2AX antibody, AW228881 antibody, H2ax antibody, Hist5-2ax antibody, gammaH2ax antibody, zgc:56329 antibody, h2a.x antibody, h2a/x antibody, h2ax antibody, RGD1566119 antibody, h2a antibody, h2afx antibody, H2A histone family member X antibody, H2A histone family, member X antibody, H2A histone family member X L homeolog antibody, histone cluster 1, H2ah antibody, histone cluster 2, H2ab S homeolog antibody, histone H2AX antibody, H2AFX antibody, H2afx antibody, h2afx antibody, h2afx.L antibody, HIST1H2AH antibody, hist2h2ab.S antibody, LOC100522201 antibody, LOC100720536 antibody

Background: Histones are highly basic proteins that complex with DNA to form chromatin. The H2AX histone (~15 kDa calculated molecular weight) is a member of the H2A histone family whose members are components of nucleosomal histone octamers. Double-stranded breaks in DNA caused by replication errors, apoptosis, or other physiological processes (including, immunoglobulin and TCR gene recombinations) and DNA damage caused by ionizing radiation, UV light, or cytotoxic agents lead to phosphorylation of H2AX on serine 139. H2AX (pS139) is also referred to as H2AX (pS140) when the N-terminal methionine that is normally excised during posttranslational processing is included in amino acid sequence numbering. Kinases such as ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) phosphorylate H2AX to induce its function. Phosphorylated H2AX (also termed, gamma-H2AX) functions to recruit and localize DNA repair proteins or cell cycle checkpoint factors to the DNA-damaged sites. In this way, phosphorylated H2AX promotes DNA repair and maintains genomic stability and thus helps prevent oncogenic transformations. Immunofluorescent staining and bioimaging analysis of cultured cells can be used to readily identify H2AX (pS139)-containing foci. As such, H2AX (pS139) immunofluorescence localization serves as a biomarker for nuclear sites of DNA damage (e.g., double-stranded DNA breaks) in affected cells.
Synonyms: H2A.X, H2A/X, H2AFX, HIST5-2AX, gamma-H2AX, gamma-H2AX, H2AX (pS140)

Pathways: Telomere Maintenance, DNA Damage Repair, Positive Regulation of Response to DNA Damage Stimulus