Methods of biochemcial analysis by David Glick

By David Glick

An encyclopaedia of tools in biochemical research, this contemporary sequence retains biochemists and analytical chemists abreast of experimental techniques and enhancements in biochemical ideas and instrumentation.

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1982), Coomassie brilliant blue (Burnette, 1981)]. Staining is followed by destaining in the same solvent minus the dye. Amido black tends to be rather sensitive [a 25 ng dot of bovine serum albumin (BSA) is easily detected] and acceptable background can be achieved. Coomassie brilliant blue tends to give higher background; Ponceau S gives very clean patterns, but it is slightly less sensitive than Amido black. None of these dyes can be used for nylon-based membranes because the background is as high as the signals and destaining is not possible.

In contrast, histochemical analyses are less hazardous, but overdevelopment of signals cannot be corrected. The resolution of enzyme-linked assays appears to be superior to that of autoradiography. XII. CORRELATING A SIGNAL TO A BAND The final result of a blot experiment is often a single band somewhere in an otherwise blank X-ray film or filter. It then becomes necessary to ascribe the signal to a particular lane of the gel and, more specifically, to a discrete polypeptide within the electrophoretic pattern.

A positive control can serve as an efficient internal standard; for example, ovalbumin serves as a convenient positive control in concanavalin A overlay experiments. Thus if one has a reliable sample whose signal is well characterized, new signals can be compared with it. When possible, probing stained filters is extremely useful. In this respect transient staining can be of help. A blot is stained with Ponceau S and destained with water (see Section VIII). The place of specific bands is marked with a pen and destaining is continued until all the Ponceau S has been removed.

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