Cross-validation of Antibodies Using DigiWest

Here, we present a novel approach that combines the principles of the western blot (protein separation by SDS-PAGE; protein immobilization on a solid support; detection by specific antibodies with a multiplexed bead array as a readout system. The system is able to generate of hundreds of bead-based western blot equivalents from a few micrograms of protein, allowing the user to obtain information on the expression and modification of hundreds of proteins from a small sample. This new approach is highly reproducible and has both good linearity and a large dynamic range.
Primary antibodies are the most critical component of both conventional western blots and DigiWest. Therefore, good antibody validation dedicated to each application is key to good and reliable results. During this webinar we will show what a good antibody validation protocol means and how this differs in different applications.

Taking advantage of the capabilities of DigiWest, we performed comprehensive signal transduction analysis of a lung cancer cell line that was rendered resistant to the kinase inhibitor lapatinib. To analyze the activation state of different signaling cascades hundreds of antibodies were employed while only micrograms of protein were required. Major changes in the activation state of Ephrin-mediated signaling and a central role for p53-controlled processes that form the basis of the observed resistance could be revealed.