For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
All Species Expected
Monoclonal antibody is produced by immunizing animals with synthetic phospho-ATM/ATR substrate peptides.
Ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) are related kinases that regulate cell cycle checkpoints and DNA repair (1). The identified substrates for ATM are p53, p95/NBS1, MDM2, Chk2, BRCA1, CtIP, 4E-BP1, and Chk1 (1,2) The essential requirement for the substrates of ATM/ATR is S*/T*Q. Hydrophobic amino acids at positions -3 and -1, and negatively charged amino acids at position +1 are positive determinants for substrate recognition by these kinases. Positively charged residues surrounding the S*/T*Q are negative determinants for substrate phosphorylation (3). The complex phenotype of AT cells suggests that it likely has additional substrates (3). To better understand the kinase and identify substrates for ATM and the related kinase ATR, CST has developed antibodies that recognize phosphorylated serine or threonine in the S*/T*Q motif.
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