A Ras-Based Module to Generate 32P-Labeled Fusion Proteins for Blot Overlays

A variety of techniques can be used to find protein partners, including immunoprecipitation, affinity chromatography, blot overlays, and yeast twohybrid screening. Of these, the blot overlay protocol of sodium dodecyl sulfate (SDS)-acrylamide-gel-separated proteins provides perhaps the most direct assessment of target binding, since one can simultaneously screen multiple cell types or tissue extracts, and infer the size-and in some cases the relative affinity-of binding proteins. Combined use with other techniques such as protein fractionation and affinity chromatography, blot overlay allows us to trace target proteins and undertake their purification. Overlays have also been successfully used for screening of expression libraries (see refs. 1 ,2 , yet the success of the yeast two-hybrid system (3 ) has partially eclipsed the use of this method for cDNA screening.