Better performance of Western blotting:quick vs slow protein transfer,blotting membranes and the visualization methods

Objective To study how the choices of the quick vs slow protein transfer,the blotting membranes and the visualization methods influence the performance of Western blotting. Methods The cellular proteins were abstracted from human breast cell line MDA-MB-231 for analysis with Western blotting using quick (2 h) and slow (overnight) protein transfer,different blotting membranes (nitrocellulose,PVDF and nylon membranes) and different visualization methods (ECL and DAB). Results In Western blotting with slow and quick protein transfer,the prestained marker presented more distinct bands on nitrocellulose membrane than on the nylon and PVDF membranes,and the latter also showed clear bands on the back of the membrane to very likely cause confusion,which did not occur with nitrocellulose membrane. PVDF membrane allowed slightly clearer visualization of the proteins with DAB method as compared with nitrocellulose and nylon membranes,and on the latter two membranes,quick protein transfer was likely to result in somehow irregular bands in comparison with slow protein transfer. With slow protein transfer and chemiluminescence for visualization,all the 3 membranes showed clear background,while with quick protein transfer,nylon membrane gave rise to obvious background noise but the other two membranes did not. Conclusions Different membranes should be selected for immunoblotting according to the actual needs of the experiment. Slow transfer of the proteins onto the membranes often has better effect than quick transfer,and enhanced chemiluminescence is superior to DAB for protein visualization and allows highly specific and sensitive analysis of the protein expressions.