Improving precision in gel electrophoresis by stepwisely decreasing variance components.
Many methods have been developed in order to increase selectivity and sensitivity in proteome research. However, gel electrophoresis (GE) which is one of the major techniques in this area, is still known for its often unsatisfactory precision. Percental relative standard deviations (RSD%) up to 60% have been reported. In this case the improvement of precision and sensitivity is absolutely essential, particularly for the quality control of biopharmaceuticals. Our work reflects the remarkable and completely irregular changes of the background signal from gel to gel. This irregularity was identified as one of the governing error sources. These background changes can be strongly reduced by using a signal detection in the near-infrared (NIR) range. This particular detection method provides the most sensitive approach for conventional CCB (Colloidal Coomassie Blue) stained gels, which is reflected in a total error of just 5% (RSD%). In order to further investigate variance components in GE, an experimental Plackett-Burman screening design was performed. The influence of seven potential factors on the precision was investigated using 10 proteins with different properties analyzed by NIR detection. The results emphasized the individuality of the proteins. Completely different factors were identified to be significant for each protein. However, out of seven investigated parameters, just four showed a significant effect on some proteins, namely the parameters of: destaining time, staining temperature, changes of detergent additives (SDS and LDS) in the sample buffer, and the age of the gels. As a result, precision can only be improved individually for each protein or protein classes. Further understanding of the unique properties of proteins should enable us to improve the precision in gel electrophoresis.
Schröder
Simone
S
Institute of Pharmaceutical Chemistry, TU Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany. si.schroeder@tu-bs.de
Brandmüller
Asita
A
Deng
Xi
X
Ahmed
Aftab
A
Wätzig
Hermann
H
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods* / Q000592:standards; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods
D011506:Proteins