| Assay Method Information | |
| | PIM1 Activity Assay |
| Description: | Dowex Resin Preparation 500 g of wet resin (SIGMA, custom prepared resin DOWEX 1x8 200-400 mesh, 2.5 Kg) are weighed out and diluted to 2 L in 150 mM sodium formate, pH 3.00.The resin is allowed to settle down (some hours) and then the supernatant is discarded.After three washes as above over a couple of days, the resin is allowed to settle and two volumes (wrt the resin volume) of 150 mM sodium formate buffer are added.The pH is then measured and should be around 3.00.The washed resin is stable for more than one week; the stock resin is kept at 4° C. before use. ii. Kinase Buffer (KB)The buffer for PIM1 assay was composed of HEPES 50 mM, at pH 7.5, with 10 mM MgCl2, 1 mM DTT, 3 uM NaVO3, and 0.2 mg/mL BSA.Full-length human PIM1 was expressed and purified as described in Bullock A N, et al., J. Biol. Chem. 2005, 280, 41675-82.The enzyme showed a linear kinetic after a step of pre-activation by auto-phosphorylation in the following conditions: 1.7 uM PIM1 was incubated 1 h at 28° C. in the presence of 125 uM ATP. |
| Affinity data for this assay | |
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