Diffusion blotting for rapid production of multiple identical imprints from sodium dodecyl sulfate polyacrylamide gel electrophoresis on a solid support.
A simple and fast method for diffusion blotting of proteins from precast SDS-PAGE gels (0.5 mm) was developed. The efficiency of protein transfer was evaluated using 14C labelled proteins. Diffusion blotting for three minutes, gave a transfer of 10% compared to electroblotting. By doubling the transfer time for each subsequent imprint, four imprints were made from the same lane with similar amounts of protein transferred onto each imprint. With a transfer time of three minutes each, it was possible to obtain at least ten imprints with all the proteins visible in all the imprints. There was no detectable loss in resolution as compared to electroblotting. The method also works well with an immuno-detecting system. The number of imprints which can be obtained, is dependent on the sensitivity of the detection system and the amount of protein applied. The greatest advantage of diffusion blotting compared to electroblotting is that several imprints can be made from each lane, and different antisera can be tested on identical imprints. The gel remains on its plastic support which prevents it from stretching and compression; this ensures identical imprints and facilitates more reliable molecular mass determination. If only a few imprints are made, sufficient protein remains within the gel for general protein staining. These advantages make diffusion blotting the method of choice when quantitative protein transfer is not required.
Olsen
I
I
Institute of Immunology and Rheumatology, The National Hospital, Oslo, Norway. ingrid.olsen@vetinst.no
Wiker
H G
HG
D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification; D002417:Cattle; D003101:Collodion; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008567:Membranes, Artificial; D008970:Molecular Weight; D009162:Mycobacterium avium / Q000737:chemistry; D010969:Plastics; D011506:Proteins / Q000302:isolation & purification*; D011817:Rabbits; D012680:Sensitivity and Specificity; D013997:Time Factors
D001426:Bacterial Proteins; D008567:Membranes, Artificial; D010969:Plastics; D011506:Proteins; D003101:Collodion