Phosphorylation assay

GMCSF)-stimulated STAT5 phosphorylation assay in TF1 cells. The kinase selectivity of these compounds was reproduced functionally in a JAK3-dri-ven, IL-2-stimulated STAT5 phosphorylation assay in HT2 cells. Both examples demonstrated favorable iv pharmacokinetics in Sprague-Dawley rats [42]. 

While imatinib is efficacious in treating Philadelphia chromosome positive CML, it is not as effective a treatment for patients with Philadelphia chromosome positive ALL [47]. As a prelude to clinical investigation, AMN107 was studied for its effect on Bcr-Abl positive ALL cell lines [48]. In prohferation and Bcr-Abl phosphorylation assays with two patient-derived ALL cell hnes, namely Z-119 and Z-181, AMN107 was 30-40-fold more potent than imatinib. 

In other work, using a range of different protein function microarrays, each fabricated in the form of BCCP fusion proteins as described here, we have been able to monitor a wide range of protein activities, including protein-protein interactions and protein-small molecule interactions, as well as carrying out on-chip phosphorylation assays (6) (see Note 15). 

In the phosphorylation assay, we used 33P-ATP to radiolabel those elements of the array that were substrates for the relevant kinase the arrays were then read using a phosphorimager (6). We have also shown that by manipulating the experimental conditions in array-based protein-protein interaction assays, we can readily distinguish true from false positives. For example, we studied the interaction of calmodulin with a diverse array of human proteins in the presence and absence of calcium (2+) ions. Because calmodulin binding should be calcium dependent, we were able to deduce true from false positives based on the array data (Fig. 11) (unpublished data). 

In our experience, the best support for cell-binding assays is a plastic slide. PVDF membrane has high adsorption capacity, and microarrays printed on such membrane are suitable for enzyme-linked colorimetric and phosphorylation assays. However, researchers are encouraged to try all of them for their specific applications. 

In a functional phosphorylation assay [y33P]-ATP and a specific protein kinase were used to label peptide substrate spots. 

Figure 18. Molecular sieve HPLC examination of CrATP-induced Ca2+ occlusion in mutants of the SR Ca2+-ATPase. The Ca2+ occluded enzyme was formed by incubation with 10 pM45Ca2+ and CrATP. The enzyme was solubilized by non-ionic detergent and injected into an HPLC column. Absorbance of the eluate was read continuously at 226 nm ( - ), and fractions were collected for analysis of radioactivity (o) or immunoreactivity specific for the Ca2+-ATPase (o). The right panel shows the absence of occluded 45Ca2+ from the Ca2+-ATPase protein peak corresponding to a mutant with severely reduced apparent Ca2+ affinity in the phosphorylation assay (c.f., Figure 17). The left panel shows as control a peak of 45Ca2+ associated with a mutant that binds Ca2+ normally.

Fig 2. Phosphorylation of histone and autophosphorylation as a function of added cyt.be/f complex. The complex was incubated under phosphorylation assay conditions (2) with (lower panel) or without added histone (upper panel), followed by SDS-PAGE, and autoradiography. 

Fig. 10. Array-based phosphorylation assay using an exogenous kinase. An array of 96 human protein kinases was printed in dupiicate and assayed in the presence and absence of exogenous FES kinase, (a) Schematic of assay, (b) 100 p.M ATP pius kinase buffer oniy. (c) 100 p,M ATP, kinase buffer pius exogenous FES kinase. The assays were deveioped using a fiuorescentiy iabeiied anti-phosphotyrosine antibody and reveaied a number of substrates for FES kinase, including MKNK1, STK6, and STK25, as marked.

EGFR-mediated intracellular tyrosine phosphorylation assay. Not determined. 

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