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[交流]
【交流/求助】关于MAPK蛋白激酶的活性测定
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各位同仁大家好： 我查阅了很多关于植物MAPK蛋白激酶的活性测定方式，但是，绝大部分都是利用同位素的方法，我想问一下，大家有没有什么好的意见或者参考方法。非常迫切期待你们的帮助。不胜感激。该激酶又名：植物促分裂素活化蛋白激酶。 祝大家身体健康，万事如意。 [ Last edited by amisking on 2009-12-22 at 21:18 ] |
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，没有做过的前辈吗？
2楼2009-03-03 08:18:07
youren2008
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新人小玥
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rkrl001(金币+3,VIP+0):很有启发，谢谢指点！ 12-23 19:46
rkrl001(金币+3,VIP+0):很有启发，谢谢指点！ 12-23 19:46
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MAPK activity assay 不知道你是要做哪种MAPK， ERK1/2, p38, JNK这几种之中的哪一种？ Intracellular Kinases Protein kinases transfer phosphate groups from ATP to serine, threonine, or tyrosine residues on protein peptide substrates, directly affecting the activity and function of the target. Radiolabel studies suggest that approximately 30% of proteins in eukaryotic cells are subject to phosphorylation. Kinase activity, a crucial post-translational modification, regulates a broad range of cellular activities including the cell cycle, differentiation, metabolism, and neuronal communication. In addition, abnormal activity of Akt, ERK, JNK, PKC, PKA, p38, and other MAPK are implicated in many disease states. R&D Systems offers a range of quality products for the detection of protein phosphorylation, which include kinase activity assays, phospho-specific antibodies, ELISA, and more. （1）ERK：ERK1/ERK2 View ERK1/ERK2 IHC images.ERK1 and ERK2 (also known as MAPK3 and MAPK1) are 44- and 42-kDa Ser/Thr kinases, respectively. They are part of the Ras-Raf-ERK signal transduction cascade often found downstream of growth factor receptor activation. ERK1 and ERK2 were initially isolated and cloned as kinases activated in response to insulin and NGF. They are expressed in most, if not all, mammalian tissues. Dual threonine and tyrosine phosphorylation activate both ERKs, at Thr202/Tyr204 for human ERK1 and Thr185/Tyr187 for human ERK2. ERK5, also known eceptor tyrosine kinases, G protein-coupled receptors, and osmotic stress. Like ERK1 and ERK2, ERK5 contains the conserved Thr-Glu-Tyr activation motif in its activation loop. Unlike these ERKs, however, ERK5 contains a unique C-terminal domain that regulates its activation and nuclear translocation （2）p38 p38 MAP Kinase The p38 Mitogen-activated Protein Kinases (MAPKs) are a family of four related Ser/Thr kinases activated by proinflammatory cytokines and environmental stresses. All four p38 family members, alpha, beta, gamma, and delta, are phosphorylated by MKK3 and/or MKK6 at dual Thr and Tyr positions within the phosphoacceptor sequence Thr-Gly-Tyr. Once activated, p38 phosphorylates a number of targets, including the nuclear transcription factors ATF2 and Max. The most frequently analyzed family member, p38 alpha, also known as SAPK2a and MAPK14, was initially purified as a kinase critical to the signaling cascade linking IL-1 to MAPKAPK-2 and the small heat shock protein HSP27. Ubiquitously expressed, p38 alpha is dually phosphorylated by MKK3 and MKK6 at Thr180 and Tyr182. Once activated, p38 alpha phosphorylates a number of targets, including the cytoplasmic kinases MNK 4 and PRAK5 and the nuclear transcription factors ATF2 1 and STAT1. Several promising compounds that inhibit p38 alpha are being investigated as potential therapies for arthritic and inflammatory diseases. (3) JNK Members of the MAPK family, the c-Jun N-terminal kinases (JNKs) are activated by environmental stresses and inflammatory cytokines. Ten JNK isoforms are created by alternative splicing of mRNA transcripts derived from three genes: JNK1, JNK2, and JNK3. All JNKs are activated by dual phosphorylation; at T183/Y185 for JNK1 and 2, and T221/Y223 for JNK3. Activated JNKs translocate to the nucleus where they regulate the activity of several transcription factors; including the c-Jun component of AP-1 and ATF-2. 在这里基本上把几种主要的MAPK的底物都列举出来了。用磷酸化的抗体跑western来检测酶的活性是其中的一种方法；用底物的磷酸化效应跑western可以进一步验证。 |
8楼2009-12-23 15:50:08
