Simultaneous Chemiluminescent and Fluorescent Detection: A Novel Approach for Double Western Blot Analysis of Low Copy Protein Targets

In this application note by Carestream Molecular Imaging, the effects of the addition of HRP substrates, on the fluorescent signal of Alexa Fluor® labelled antibodies in Western blotting are assessed. Additionally, a protocol is presented that describes both the capture of luminescent and fluorescent images, as well as post image capture processing (i.e. pseudocolor and image overlay) to produce publication quality figures. Chemiluminescent Western blot analysis remains the most sensitive method for detection of low-copy protein targets. Since most loading control proteins are highly expressed proteins that can be easily detected using fluorescence, and most researchers are trying to detect low-copy proteins of interest, a method is developed for simultaneous incubation of two protein targets using both fluorescent and chemiluminescent secondary antibodies.