Signal Transduction Protocols by Robert C. Dickson, Michael D. Mendenhall

By Robert C. Dickson, Michael D. Mendenhall

Sporting at the excessive criteria of the much-acclaimed first version, hugely skilled investigators have commonly up-to-date the 1st version with some of the new techniques which were remodeling the sector. incorporated during this new version are effectively reproducible immunoassays, fluorescence-based assays, high-throughput equipment, protein amendment assays, lipid moment messenger assays, and chromatin immunoprecipitation suggestions. at any place attainable, protocols for the assay of normal periods of sign transduction parts were pointed out to allow them to be tailored to the assay of any member of that classification.

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2. Choosing a Reporter Vector Here again there are a variety of systems to choose from. Firefly luciferase has been our reporter of choice simply because of its sensitivity and the availability of its substrate. A variety of other luminescent enzymes have been cloned more recently along with their available substrates (13–15). In addition there are a variety of fluorescing proteins that have been, or can be, adapted to function as reporters (15–17). Use of these in high-throughput assays will require a plate-reading fluorimeter, but might be worth the investment if one wishes to develop scenarios that incorporate some form of fluorescence energy transfer (FRET) into their readout (9,17).

Biochemistry 41, 1070–1078. 2-D Electrophoresis for Signaling Analysis 49 5. , et al. (2000) The Fas-induced apoptosis analyzed by high throughput proteome analysis. J. Biol. Chem. 275, 39,018–39,026. 6. , et al. (1999) Defining protease specificity with proteomics: a protease with a dibasic amino acid recognition motif is regulated by a two-component signal transduction system in Salmonella. Electrophoresis 11, 2241–2247. 7. , et al. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradient.

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