| Assay Method Information | |
| | Radioactive Kinase Assay |
| Description: | The ATP-KM value of the complex is 61 uM. The kinase assays are run in the presence of 100 uM ATP using 10 uM of a substrate peptide. pAUB-IN847 was used to transform the E. coli strain BL21 (DE3) containing the pUBS520 helper plasmid. Both proteins and their mutants are expressed and purified under essentially identical conditions. Protein expression is induced with 0.3 mM IPTG at an OD6oo of 0.45-0.7. Expression is then continued for about 12-16 hours at 23-25 C with agitation. Bacterial cells are harvested by centrifugation at 4000 rpm x 15 min in a Beckman JLA 8.1 rotor, and the pellets resuspended in lysis buffer (50 mM Tris HCI pH 7.6, 300 mM NaCI, 1 mM DTT, 1 mM EDTA, 5 % glycerol, Roche Complete protease inhibitor tablets). 20-30 ml lysis buffer are used per liter of E. coli culture. Cells are lysed by sonication, and the lysates cleared by centrifugation at 12000 rpm for 45-60 min on a JA20 rotor. |
| Affinity data for this assay | |
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