Visible fluorescent detection of proteins in polyacrylamide gels without staining.
2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.
Ladner
Carol L
CL
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. N.W., Calgary, Alta., Canada, T2N 1N4. redwards@ucalgary.ca
Yang
Jing
J
Turner
Raymond J
RJ
Edwards
Robert A
RA
D000180:Acrylic Resins; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005023:Ethylene Chlorohydrin / Q000031:analogs & derivatives*; D005453:Fluorescence; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012394:Rosaniline Dyes; D013194:Staining and Labeling / Q000379:methods*; D014238:Trichloroacetic Acid
D000180:Acrylic Resins; D011506:Proteins; D012394:Rosaniline Dyes; C016680:polyacrylamide gels; D014238:Trichloroacetic Acid; D005023:Ethylene Chlorohydrin; C005849:2,2,2-trichloroethanol; C004692:coomassie Brilliant Blue