A fast Western blot procedure improved for quantitative analysis by direct fluorescence labeling of primary...

The procedures for Western blots have been around for a long time and recent developments have increased the sensitivity for luminescent techniques so that the need for radioactive probes has been limited to only a few applications. Nevertheless, most protocols require more than 6 h and are often performed over more than a day. The majority of techniques require a secondary antibody conjugated to an enzyme that catalyzes a color reaction in order to amplify a detectable signal. However, both processes, the binding of a secondary antibody and the catalyzed reaction with the dye, are sources for errors and the latter is disadvantageous for a signal that is linear over a larger range of detected antigen. In order to improve the procedure most commonly used for quantitative analysis and convenience, we investigated the use of fluorescence labeling of primary monoclonal antibodies against Escherichia coli RNA polymerase subunits (beta', sigmaE and sigmaFecI) and their use in Western blots. We achieved a sensitivity (<1 ng detectable protein) comparable to most luminescent techniques. Additionally, we reduced the procedure time significantly to less than 1 h after SDS-PAGE and transfer to a membrane. Above all, we obtained a linear signal over the range of 30 ng to 1 microg of protein (dependent on protein size) making quantitative analysis of Western blots easier and more reliable.