A solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters☆
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Cited by (18)
Profiling substrate phosphorylation at the phosphopeptide level
2003, Analytical BiochemistryCitation Excerpt :The high degree of resolution and reproducibility by which 2-D profiles of protein digests are generated by this experimental system, combined with the possibility of identifying the fractionated phosphopeptides, prompted me to use this peptide mapping system to profile the kinase activity of γ-PAK on unknown substrates. The idea of using a solid-phase kinase assay to profile substrate phosphorylation was based on the seminal report of Valtorta et al. [3], according to which the patterns of protein phosphorylation resulting from the use of nitrocellulose-immobilized substrates are similar to those obtained with the conventional liquid-phase kinase assay [3]. In the present study, the respective sites of phosphorylation of MBP (Figs. 1 and 2) and H4 (data not shown) were found to be equally targeted upon solid- and liquid-phase kinase assay, thus ruling out the possibility of an artifactual phosphorylation of nitrocellulose-immobilized substrates.
Dynamin is a minibrain kinase/dual specificity Yak1-related kinase 1A substrate
2002, Journal of Biological ChemistryCitation Excerpt :A rapid and reliable assay is essential for the purification of kinase substrates. Therefore, a solid-phase kinase assay was developed for Mnbk/Dyrk1A by adapting the combination of two previously described protocols (36, 39). Briefly, proteins to be tested are separated by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, blocked by bovine serum albumin, and then subjected to Mnbk/Dyrk1A phosphorylation in the presence of [γ-32P]ATP.
A solid-phase screen for protein kinase substrate selectivity
1992, Analytical BiochemistryEnzyme activity dot blots for assaying protein kinases
1991, Methods in EnzymologyChapter 12 Regulation of retinal functions by octopaminergic efferent neurons in Limulus
1991, Progress in Retinal Research
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This work was supported by the United States Environmental Protection Agency under Assistance Agreement CR-810608-01 (P.G.) and by a M.D.A. grant to B.C.