| Assay Method Information | |
| | In Vitro Enzyme Assay |
| Description: | ATR for use in the in vitro enzyme assay was obtained from HeLa nuclear extract (CIL Biotech, Mons, Belgium) by immunoprecipitation with rabbit polyclonal antiserum raised to amino acids 400-480 of ATR (Tibbetts R S et al, 1999, Genes Dev. 13:152-157) contained in the following buffer (25 mM HEPES (pH7.4), 2 mM MgCl2, 250 mM NaCl, 0.5 mM EDTA, 0.1 mM Na3V04, 10% v/v glycerol, and 0.01% v/v Tween 20). ATR-antibody complexes were isolated from nuclear extract by incubating with protein A-Sepharose beads (Sigma, #P3476) for 1 hour and then through centrifugation to recover the beads. In the well of a 96-well plate, 10 ATR-containing Sepharose beads were incubated with 1 μg of substrate glutathione S-transferase-p53N66 (NH2-terminal 66 amino acids of p53 fused to glutathione {circumflex over ( )}-transferase was expressed in E. coli) in ATR assay buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 6 mM MgCl2, 4 mM MnCl2, 0.1 mM Na3V04, 0.1 mM DTT, and 10% (v/v) glycerol) at 37° C. in the presence or absence of inhibitor. After 10 minutes with gentle shaking, ATP was added to a final concentration of 3 μM and the reaction continued at 37° C. for an additional 1 hour. The reaction was stopped by addition of IOOμ PBS and the reaction was transferred to a white opaque glutathione coated 96-well plate (NUNC #436033) and incubated overnight at 4° C. This plate was then washed with PBS/0.05%>(v/v) Tween 20, blotted dry, and analyzed by a standard ELISA (Enzyme-Linked Immunosorbent Assay) technique with a phospho-serine 15 p53 (16G78) antibody (Cell Signaling Technology, #9286). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |