MAP Kinase Signaling Protocols by Rony Seger

By Rony Seger

An excellent selection of either vintage and state-of-the-art ideas for the detection and size of mitogen-activated protein kinase (MAPK) signaling cascades. The protocols contains tools for the decision of the subcellular localization of those parts, the structural and biophysical research of the elements, and identity of novel elements of recognized and unknown signaling cascades. extra equipment research the upstream mechanisms of activation of MAPK cascades by way of numerous receptors, the mechanisms focused on the down rules of the MAPK cascades, and identity of ambitions of the MAPK cascades. a few innovations use inhibitors in learn on MAPK and transgenic mice for experiences of MAPK signaling. The suggestions supply biochemists, telephone biologists, physicians, and biotechnologists all of the crucial laboratory recommendations wanted in to behavior effective experiences of MAPK signaling in healthiness and ailment, in measuring the impact of gear, and in a wide variety of experimental structures.

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The AP detection system, which has a moderate linear range, is usually used for the types of experiments described here, because it is a convenient method. Immunoprecipitation methods vary in the order in which the antibodies and protein A are added to the cell extracts. In the protocol described here, the antibodies are conjugated to protein A beads, and only then added to the cytosolic extracts. This procedure minimizes the time of incubation of the samples with the antibodies, and thereby minimizes exposure of the desired kinases to phosphatases and proteinases in the extracts.

Blocking solution: 2% (w/v) BSA in washing buffer. , monoclonal antidiphospho-ERK [M-8159] and polyclonal anti–general ERK [M-5670] from Sigma, Israel), and secondary antibody (alkaline phosphatase [AP]–or horseradish peroxidase [HRP]–conjugated antimouse or antirabbit Fab antibodies from Jackson) diluted in washing buffer to appropriate dilutions. Enhanced chemiluminescence (ECL): Commercial kits are available from Amersham, Pierce, and Bio-Rad). 5). 0 mM MgCl 2) containing 66 µL of NBT (50 mg/mL) (#S380C; Promega) and 33 µL of BCIP (50 mg/mL) (#S381C; Promega).

Et al. (2000) Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases. FEBS Lett 468, 37–42. 20. , Bonventre, J. , and Kyriakis, J. M. (1994) Enzymatic characteristics of the c-Raf-1 protein kinase. Proc. Natl. Acad. Sci. USA 91, 1270–1274. 21. , Diltz, C. , and Fischer, E. H. (1996) Activation of mitogen-activated protein (MAP) kinase pathway by pervanadate, a potent inhibitor of tyrosine phosphatases. J. Biol. Chem.

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