Essential problems in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels, and their solution.
Quantitative determination of stained proteins following polyacrylamide gel electrophoresis (PAGE) is of increasing interest especially since computer-aided densitometers have become available as well as recipes for sensitive and background-free staining with Coomassie Brilliant Blue dyes. However, avoidance of separation artifacts is not the only essential prerequisite for quantitative evaluation. The local particle density of a protein in a given gel is of critical importance since it determines its stainability. Depending on local protein concentration, the dye binding to the same amount of a given protein differs considerably. Since the stainability of proteins using colloidal staining procedures, as with Coomassie Brilliant Blue dyes, is time-dependent and, in addition, also dependent on the pore size of a given polyacrylamide gel used for PAGE, calibration curves for quantitative determinations have to be prepared in polyacrylamide gels of the same composition as used for PAGE. Staining conditions also have to be identical for calibration gels and gels under analysis. If, however, a set of calibration curves is prepared for different staining times, it is possible to calculate a generalized calibration curve, allowing for quantitative evaluation with flexible staining time. Furthermore, and in consequence of the implications due to particle density, quantitative determination via densitometry is only possible by determining the protein amount of each single measuring point (pixel) via its absorbance on the basis of a calibration curve. Since the particle density is inherent in a calibration curve, the final summation of the protein amount per pixel will give values close to reality.
Max-Planck-Institut für experimentelle Medizin, Göttingen, Federal Republic of Germany.
V
V
Neuhoff
R
R
Stamm
I
I
Pardowitz
N
N
Arold
W
W
Ehrhardt
D
D
Taube
D002138:Calibration; D003102:Colloids; D003720:Densitometry; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel; D008027:Light; D008956:Models, Chemical; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012394:Rosaniline Dyes; D012542:Scattering, Radiation; D013194:Staining and Labeling
D003102:Colloids; D011506:Proteins; D012394:Rosaniline Dyes; C004692:coomassie Brilliant Blue