Simultaneous Immunoblotting Analysis with Activity Gel Electrophoresis

Abstract

AbstractTransferring proteins partially from a gel to a membrane by diffusion blotting is useful to (a) probe a protein on the membrane with an antibody and (b) allows one to study the gel by the method of zymography. Thus one can use a single original SDS polyacrylamide gel to combine immunoblotting analysis with another biochemical technique. However, one needs to note that there will be lower transfer efficiency than the traditional methods of transferring proteins to the membrane using electric current. Thus, this procedure, originally developed and described by Lee and Chang, allows protein blots from: 1. An SDS polyacrylamide gel, which one can then use for zymography assay 2. A native polyacrylamide gel for a method known as electrophoretic mobility shift assay (EMSA) 3. A two-dimensional polyacrylamide gel for large-scale screening and identification of a protein marker The proteins transferred to the membrane in each case can be identified for a specific signal in zymography, EMSA, or two-dimensional gels by simultaneous immunoblotting analysis with a corresponding antibody. The advantages listed above make diffusion blotting attractive when a fractional loss of transfer efficacy can be tolerated or recompensed using a more sensitive immunodetection (e.g., enhanced chemiluminescence detection).KeywordsDiffusionProtein transferBlottingZymographyEMSAElectrophoretic mobility shift assayAntibody