The protocol is a straightforward adaptation of conventional Western blotting. The sample proteins are first electrophoretically separated on a gel then transferred from the gel onto the stack of 3, 5 or 10 membranes using the transfer buffer. Electro transfer is accomplished using conventional Western blotting apparatus. Each of the blots can then be probed with a different antibody. Immunodetection is performed with either chemiluminescent or colorimetric substrates. The membranes are then digitalized using either a flatbed scanner or one of several standard imaging instruments used with gel blots. The images generated by the various layers can then be analyzed using special software currently in development. Among the features of the software will be densitometry analysis to compare expression levels of various targets to one another. 

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