SDS-PAGE of proteins using a chameleon-type of fluorescent prestain.
A new prestaining method for protein SDS-PAGE was developed using the fluorogenic amino-reactive label Py-1. This resulted in one of the fastest, most sensitive, and environmentally friendly protocols available. It is mainly due to the unique optical properties of Py-1, which is blue and virtually nonfluorescent but turns to red and becomes much more strongly fluorescent once it is conjugated to the amino group of a protein. Staining times of 30 min are adequate to visualize subnanogram quantities of proteins because pre-electrophoretic labeling Py-1 does not require the time-consuming steps of washing or fixation of gels. LODs as low as 16 pg of protein are found which is better than the best (commercial) poststains and comparable to the best (commercial) prestains. In addition, prestaining requires marginal amounts of staining solution. The change in electrophoretic mobility and band broadening is at a low level because Py-1 causes a mass shift of 288 Da per bound molecule only. By virtue of the small mass shift it causes, this stain is compatible with mass spectrometric protein analysis even though it acts as a covalent label.
Meier
Robert J
RJ
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany.
Steiner
Mark-Steven
MS
Duerkop
Axel
A
Wolfbeis
Otto S
OS
D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D011506:Proteins