Protocol for: Increased Detection Sensitivity for Chemiluminescent and Fluorescent Western Blotting Using Signal Enhancer and
Abstract or Description:
Whether a researcher is utilizing chemiluminescent or fluorescent Westerns, he or she wants to be able to detect the protein of interest on the first try. However, there are many variables that come into play when performing a Western blot, such as membrane type, blocker, copy number of the target protein present in the sample, primary and secondary antibody concentration, antibody affinities, and detection reagents. Optimization of each variable can greatly improve detection sensitivity for a given Western blotting system. One way to determine if a Western system is working is to complete a few dot blots prior to running and transferring the gel. This sandwich assay is a dilution of the protein sample or primary antibody directly dotted onto a dry nitrocellulose (NC) membrane and incubated like a regular Western to test the amount of protein sample needed, effectiveness of the blocking agent, primary antibody affinity to the protein sample, and primary-secondary antibody affinity. The time taken to complete a simple set of dot blots can prevent the loss of valuable protein samples and expensive Western reagents. This application note addresses several of these parameters and describes ways in which a researcher can modify his or her technique in order to increase detection sensitivity.