Western blotting analysis of proteins separated by agarose native gel electrophoresis

Abstract

Western blotting was attempted to analyze proteins separated by agarose native gel electrophoresis that was previously developed on His/Mes buffer system. This report shows a simple protocol for blotting agarose native gel to a PVDF membrane by soaking the gel in sodium dodecylsulfate-containing transfer buffer and 3 examples of such analysis. First example showed expression of a recombinant antibody in HEK293 cells by direct staining of the agarose native gels for both proteins and nucleic acids and staining of the blots for proteins and host cell proteins. These analyses demonstrated usefulness of agarose native gel electrophoresis, confirming that the recombinant antibody migrates toward the cathode while nucleic acids and a majority of host cell proteins migrate toward the anode. Second example demonstrated the phosphorylation state of MAP kinase in human lymphocyte cell line. Namely, agarose native gel can separate kinase, whose phosphorylation can be analyzed by Western blotting. Third example showed correlation of Escherichia coli β-galactosidase expression between the oligomerization and enzyme activity using antibody and substrate staining.

Keywords: Agarose; His/Mes buffer; Kinase; Native gel; Substrate staining; Western blotting.