γ 32p atp 5 mci

All biochemicals, including bicine, LB, metal salts and antibiotics were purchased from either Melford or Sigma and were of analytical quality. [γ⁻³²P]ATP (5 mCi) was purchased from Perkin-Elmer and diluted appropriately with unlabelled ATP. Sypro Orange dye was from Invitrogen and was diluted as described in the Invitrogen TSA protocol. A small compound library composed of 30 modified PP1 (pyrazolo-pyrimidine) analogues was prepared as previously described [45 (link)]. The compounds contain bulky additions at the C5 position of the pyrimidine ring that are designed to interact with the hydrophobic pocket adjacent to the gatekeeper residue of AS protein kinases. Compounds were stored at 10 mM in DMSO at −80°C until required and diluted directly into the assay at 250 μM, so that the final DMSO concentration was no higher than 2.5% (v/v) in any assay. Acronyms for compounds A, B and F are dClB-PP1 (A): dichlorobenzyl-PP1; dmB-PP1 (B): dimethylbenzyl-PP1; bisPh-PP1 (F): bisphenyl-PP1. Similar data were obtained from two independent compound preparations.