Immunoblotting 2DE membranes.

Abstract

Two-dimensional electrophoresis (2DE) immunoblotting combines the resolving power of 2DE and the selectivity of antibodies, allowing the selection of individual or sets of proteins from the total proteome. It is essential when immunoblotting membranes that reproducible 2DE gels are obtained consistently. Afterwards, it is a matter of applying the same principles as 1DE immunoblotting. However, it should be noted that the most sensitive methods of detection should be used. This chapter outlines some of the considerations that should be taken into account, ranging from the choice and type of antibodies, buffers involved, and methods of detection and visualisation.