Bacterial Fractionation and Membrane Protein Characterization

This chapter describes some basic procedures for the fractionation, identification, and characterization of certain bacterial envelope proteins. It argues that basic bacterial fractionation is quite uncomplicated. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) provides exceedingly good separation of bacterial component proteins, and "preparative" SDS-PAGE can often provide sufficient material for preparing high titer antisera to bacterial components. The chapter also describes a method for extracting individual proteins from SDS-PAGE gels for the purpose of preparing specific antibodies. Certain bacterial membrane proteins have distinct biochemical properties that can be exploited for the purpose of their identification and characterization. Certain membrane proteins, particularly porins, are characterized by being noncovalently linked to the peptidoglycan layer. Certain procedures used for bacterial fractionation may co-isolate membrane-associated protein structures such as flagella, fimbriae, and extracellular protein layers. These proteins will appear on SDS-PAGE gels as bands which might be mistakenly identified as, for example, outer-membrane proteins.