Figure 1: Phospho-Histone H2A.X (Ser139) protein from untreated (-) and UV-treated (+) 293 cells is detected by PathScan® Phospho-Histone H2A.X (Ser139) Sandwich ELISA kit. A lower signal of phospho-Histone H2A.X protein is seen in the untreated lysate. However, similar levels of total Histone H2A.X protein from both untreated and treated lysates are shown by western analysis. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blot using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718 is shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and UV-treated 293 cells and the absorbance at 450 nm as detected by the PathScan® Phospho-Histone H2A.X Ser139 Sandwich ELISA Kit is shown.
Unstarved 293 cells (80-90% confluence) were treated with 100 mJ/cm2 UV with 1 hr recovery at 37ºC, and then lysed with Cell Lysis Buffer (#9803).
|Product Includes||Volume||Solution Color|
|Histone H2A.X Rabbit mAb Coated Microwells||96 tests|
|P-Histone H2A.X (Ser139) Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml||Colorless|
|STOP Solution 7002||11 ml||Colorless|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml||Yellowish|
The PathScan® Phospho-Histone H2A.X (Ser139) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Histone H2A.X protein phosphorylated at Ser139. A Histone H2A.X rabbit antibody has been coated onto the microwells. After incubation with cell lysates, both phospho- and non-phospho-Histone H2A.X proteins are captured by the coated antibody. Following extensive washing, a phospho-Histone H2A.X (Ser139) mouse antibody is added to detect the captured phospho-Histone H2A.X protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Histone H2A.X phosphorylated at Ser139. Antibodies in this kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Phospho-Histone H2A.X (Ser139) Sandwich ELISA Kit detects endogenous levels of Histone H2A.X protein phosphorylated at Ser139 in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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.
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