Ultrasensitive detection of unstained proteins in acrylamide gels by native UV fluorescence.
Visualization of proteins inside acrylamide and other gels usually relies on different staining methods. To omit the protein-staining procedure, we visualized unstained proteins inside acrylamide gels by laser excitation with ultraviolet (UV) light (280 nm, 35 mJ/cm2) and directly detected native UV fluorescence. In one-dimensional gels, a detection limit as low as 1 ng for bovine serum albumin and 5 ng for other proteins with a linear dynamic range (2.7 orders of magnitude) comparable to state of the art fluorescent dyes could be achieved. In addition, the application of this method to 20 microg of a whole cell lysate separated in a two-dimensional gel showed more than 600 spots. Since protein labeling always represents a serious obstacle in protein identification technologies, the working efficiency with our procedure can be considered as a significant improvement for protein visualization and reproducibility in proteomics.
Roegener
Jan
J
Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany.
Lutter
Petra
P
Reinhardt
Ralf
R
Blüggel
Martin
M
Meyer
Helmut E
HE
Anselmetti
Dario
D
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005453:Fluorescence; D005782:Gels; D007834:Lasers; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D014466:Ultraviolet Rays
D005782:Gels; D011506:Proteins