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Identification of bacteriophage T4 virion proteins by transverse pore‐gradient sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and dual amino acid labeling
Author(s) -
Ferguson Peter L.,
Coombs David H.
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181528
Subject(s) - gel electrophoresis , sodium dodecyl sulfate , polymerization , chemistry , polyacrylamide gel electrophoresis , chromatography , monomer , molecular weight size marker , sodium , methionine , electrophoresis , amino acid , polyacrylamide , gel electrophoresis of proteins , biochemistry , polymer , polymer chemistry , organic chemistry , enzyme
Abstract We have developed a horizontal N , N ′‐methylenebisacrylamide (Bis) acrylamide gradient sodium dodecyl sulfate (SDS) gel system that permits the evaluation of the purity of individual protein bands in complex mixtures. A Bis gradient gel is poured vertically and, after polymerization, reoriented horizontally. A single large sample spanning the top of the gel is then run down at right angles to the gradient. The relative mobility of a few proteins varies considerably from the rest, causing them to merge with and cross other bands as the Bis concentration changes. Band splitting revealed that several bands previously thought to represent a single species are actually comprised of comigrating proteins. Once the Bis/monomer concentration offering the best separation was identified, we sought a simple method for identifying the genetic origin of bands, since many proteins now migrated in new positions on the gel. We reasoned that if infected cells were simultaneously labeled with [ 35 S]methionine and [ 3 H]leucine and the purified virion proteins analyzed to determine their 35 S/ 3 H ratio, we could identify most proteins by comparing this ratio with one calculated from the T4 DNA sequence. Our expectations were realized, and we here report the separation and identification of all T4 virion proteins. Finally, we comment on the incorporation of various changes to the original Laemmli SDS‐polyacrylamide gel formulations that have been reported in the literature.