Preparing Whole-Cell Lysates for Immunoblotting.

Abstract

In this protocol, adherent cells are grown in tissue culture dishes until ready to be used for protein sample preparation. To analyze proteins by immunoblotting, it is necessary to bring the proteins from the sample into soluble form using the buffers, salts, and detergents compatible with the gel electrophoresis procedure. If a protein sample is being prepared for immunoblotting only, the proteins can be solubilized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, as described in this protocol, which ensures rapid and efficient extraction of most cellular proteins because of the presence of SDS, a strong anionic detergent. SDS-PAGE sample loading buffer denatures proteins and disrupts the bonds between the proteins that interact with each other. Therefore, this lysis technique can be detrimental to the applications when the native structure of the protein is essential, including enzymatic activity assays and analysis of protein-protein interactions.