Giant two-dimensional gel electrophoresis: methodological update and comparison with intermediate-format gel systems.
Two-dimensional (2-D) gel electrophoresis methods for separating complex mixtures of proteins have not changed fundamentally since their original description in the late 1970's. Nevertheless, 2-D gel resolution has improved substantially as a result of a series of incremental modifications. One of these was the development of the "giant-gel" format, using gels measuring at least 30 x 30 cm to provide the highest resolution 2-D gel system available. As originally described, this procedure has several important limitations: it requires custom-built equipment; it is expensive in terms of time, reagents, film and support matrices; and it generates gels which are difficult to manupulate, particularly for silver staining. This report describes modifications in the giant gel procedure to permit use of a commercially available gel apparatus and to obtain giant gels of improved mechanical strength suitable for silver staining. The resolution of giant gels is compared with that obtained using two systems currently being marketed for use by laboratories performing large numbers of 2-D gel analyses. The smaller format gels resolved fewer proteins, by 30-40%, compared with the giant gels. This difference in resolving power suggests that giant gels will continue to be useful in selected applications.
Howard Hughes Medical Institute Laboratories, Durham, NC.
R M
RM
Levenson
G M
GM
Anderson
J A
JA
Cohn
P J
PJ
Blackshear
D001345:Autoradiography; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification*; D011972:Receptor, Insulin; D012834:Silver; D013194:Staining and Labeling
D007328:Insulin; D010750:Phosphoproteins; D011506:Proteins; D012834:Silver; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin