Di- and Tri-Chromatic Fluorescence Detection on Western Blots

Abstract

The standard protocol for investigators striving to visualize concurrently the total protein profile and a specific antigen by immunodetection is to run replicate gels, using one for general protein staining and the other for immunoblotting. Similar procedures are often employed to visualize total protein and glycoprotein profiles by lectin blotting. However, the multiple manipulations required for electrophoresis and blotting often result in an inability to align bands or spots on the gel with bands or spots from the immunoblot. Shrinking, swelling, and other incidental side effects of electrophoresis, immunoblotting, and staining result in gel-to-gel and/or gel-to-blot variations, which make definitive band identification at best uncertain. The possibility of registration errors arising from the cited dimensional changes are compounded when addressing identification of specific proteins in two-dimensional (2-D) gel profiles, in which the immunoblot may have very few landmarks to aid in registration with the complex spot pattern.