This page describes common challenges encountered when lysing cells and extracting proteins prior to Western blotting. Total protein concentration must be determined for these cell lysates. Variables affecting each of these steps are outlined below, as each could affect the sensitivity and reproducibility of the Western blot.
The possible causes and potential remedies for challenges encountered during preparation of samples for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and optimizing electrophoresis conditions.
The possible causes and potential remedies for challenges encountered in the immunoprecipitation-Western blot technique, which consists of cell lysis, formation of the antibody-antigen (immune) complex, precipitation of the immune complexes, and analysis by Western blotting.