Western Blot

Western blotting is an immunoblotting technique used for the detection of proteins that have been separated by SDS-PAGE and transferred to a solid membrane support (typically nitrocellulose or PVDF).   Not all antibodies work well in a western blot assay, due to the fact that the protein is denatured and the epitope (antibody binding site) of the protein of interest may not be accessible. Specific screening may be required to detect antibodies that are compatible with western blotting and optimization of the assay conditions to maximize their performance.

In an effort to save time and money, MBS screens potential candidates as early in the process as possible to determine their utility as a western blot reagent. This initial screening helps to identify the best candidates to scale up for use in western blotting assays (Figure 1).

Figure 1: Seventeen hybridoma fusion products strongly positive at primary fusion ELISA screening were further evaluated to identify candidates capable of detecting the target analyte in a Western blot format (*add lane designations to final image)

Figure 1: Seventeen hybridoma fusion products strongly positive at primary fusion ELISA screening were further evaluated to identify candidates capable of detecting the target analyte in a Western blot format.

Following the identification of specific antibody clones, optimization of the conditions for western blotting is typically necessary (Figure 2).  While the screening is typically done with a purified protein, assay development would be performed with appropriate cell lysates or tissue homogenates as appropriate.

Figure 2: Western blot loading optimization using two different transfer membranes.

Figure 2: Western blot loading optimization using two different transfer membranes.