Quantitation of electrophoretically separated proteins in the submicrogram range by dye elution.
A new method for the fast, reliable and reproducible submicrogram quantitation of proteins separated by different electrophoretic techniques is presented. The method is based on a modified sensitive staining technique using Coomassie Brilliant Blue G-250 in colloidal solution combined with an optimized elution procedure of the bound dye in a 3% w/v solution of sodium dodecyl sulfate followed by photometric determination of dye concentration in the eluate. In addition a new method is provided for background correction, even suitable for gels showing strong background staining. The staining procedure allows the detection of 20 ng depending on the nature of the protein and the separation technique used. Quantitation is linear at least in the range from 50 ng to 10 micrograms and highly reproducible even under non-optimized conditions. The presented method can be applied to sodium dodecyl sulfate-electrophoresis, isoelectric focusing and two-dimensional electrophoresis.
Neumann
U
U
Institut für Medizinische Chemie, Medizinische Hochschule Hannover.
Khalaf
H
H
Rimpler
M
M
D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008832:Microchemistry; D009113:Muramidase / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine / Q000032:analysis; D013194:Staining and Labeling
D011506:Proteins; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine; D009113:Muramidase; C004692:coomassie Brilliant Blue