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45456
PhosphoPlus® Histone H2A.X (Ser139) Antibody Duet
Primary Antibodies
Antibody Duet

PhosphoPlus® Histone H2A.X (Ser139) Antibody Duet #45456

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Simple Western analysis of lysates (0.1 mg/mL) from COS-7 cells using Histone H2A.X (D17A3) XP® Rabbit mAb #7361. The virtual lane view (left) shows the target band (as indicated) and a band corresponding to Histone H2A.X (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western blot analysis of extracts from various cell lines using Histone H2A.X (D17A3) XP® Rabbit mAb.
Western blot analysis of extracts from untreated or UV-treated 293 cells, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (upper) or Histone H2A.X Antibody #2595 (lower).
Immunohistochemical analysis of paraffin-embedded human ovarian clear cell carcinoma using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb performed on the Leica BOND Rx.
Immunohistochemical analysis of paraffin-embedded mouse colon using H2A.X (D17A3) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb performed on the Leica BOND Rx.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Histone H2A.X (D17A3) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb, showing nuclear localization.
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Histone H2A.X (D17A3) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
Confocal immunofluorescent analysis of MCF7 cells using Histone H2A.X (D17A3) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Immunohistochemical analysis of paraffin-embedded human lung carcinoma untreated (left) or lambda-phosphatase-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
Flow cytometric analysis of MCF7 cells (green) using Histone H2A.X (D17A3) XP® Rabbit mAb (solid lines) or a concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Immunohistochemical analysis of paraffin-embedded HT-29 cells untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with UV (100 mJ, 2hr recovery; green) using Phospho-H2A.X (Ser139) (20E3) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® isotype control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
To Purchase # 45456
Cat. # Size Qty. Price
45456S
1 Kit

Product Includes Quantity Reactivity MW(kDa) Isotype
Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb 9718 100 µl H M R Mk 15 Rabbit IgG
Histone H2A.X (D17A3) XP® Rabbit mAb 7631 100 µl H M R Mk 15 Rabbit IgG

Product Description

PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.

Background

Histone H2A.X is a variant histone that represents approximately 10% of the total H2A histone proteins in normal human fibroblasts (1). H2A.X is required for checkpoint-mediated cell cycle arrest and DNA repair following double-stranded DNA breaks (1). DNA damage, caused by ionizing radiation, UV-light, or radiomimetic agents, results in rapid phosphorylation of H2A.X at Ser139 by PI3K-like kinases, including ATM, ATR, and DNA-PK (2,3). Within minutes following DNA damage, H2A.X is phosphorylated at Ser139 at sites of DNA damage (4). This very early event in the DNA-damage response is required for recruitment of a multitude of DNA-damage response proteins, including MDC1, NBS1, RAD50, MRE11, 53BP1, and BRCA1 (1). In addition to its role in DNA-damage repair, H2A.X is required for DNA fragmentation during apoptosis and is phosphorylated by various kinases in response to apoptotic signals. H2A.X is phosphorylated at Ser139 by DNA-PK in response to cell death receptor activation, c-Jun N-terminal Kinase (JNK1) in response to UV-A irradiation, and p38 MAPK in response to serum starvation (5-8). H2A.X is constitutively phosphorylated on Tyr142 in undamaged cells by WSTF (Williams-Beuren syndrome transcription factor) (9,10). Upon DNA damage, and concurrent with phosphorylation of Ser139, Tyr142 is dephosphorylated at sites of DNA damage by recruited EYA1 and EYA3 phosphatases (9). While phosphorylation at Ser139 facilitates the recruitment of DNA repair proteins and apoptotic proteins to sites of DNA damage, phosphorylation at Tyr142 appears to determine which set of proteins are recruited. Phosphorylation of H2A.X at Tyr142 inhibits the recruitment of DNA repair proteins and promotes binding of pro-apoptotic factors such as JNK1 (9). Mouse embryonic fibroblasts expressing only mutant H2A.X Y142F, which favors recruitment of DNA repair proteins over apoptotic proteins, show a reduced apoptotic response to ionizing radiation (9). Thus, it appears that the balance of H2A.X Tyr142 phosphorylation and dephosphorylation provides a switch mechanism to determine cell fate after DNA damage.

Limited Uses

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