Two-dimensional electrophoresis of human urinary proteins following concentration by dye precipitation.
The use of dye precipitation for concentration of proteins prior to 2-DE has been demonstrated by application to human urine. The precipitation methods (T. Marshall and K. M. Williams, Clin. Chem. 1993, 39, 2314-2318; T. Marshall et al., Electrophoresis 1995, 16, 28-31) have been adapted for two-dimensional electrophoresis (2-DE) by modifying the reagent composition and adding sodium dodecyl sulphate (SDS) to the assay mixture. These modifications extend the precipitation range of the methods and overcome the problem of soluble glycoprotein-dye complexes. The protein-dye complex is recovered by centrifugation and dissolved in a small volume of sample denaturing buffer. The dye separates from the protein on 2-DE (forming a sharp band at the anodal end of the isoelectric focusing gel) so that the positional coordinates of the polypeptides are unaffected by the treatment. Dye precipitation provides a simple, rapid and highly economic method for concentrating urines of low to intermediate protein content (0.02-0.50 g/L) prior to 2-DE analysis.
Marshall
T
T
School of Health Sciences, University of Sunderland, Great Britain.
Williams
K
K
D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D004396:Coloring Agents; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D011507:Proteinuria / Q000652:urine*; D012394:Rosaniline Dyes; D016622:Silver Staining
D004396:Coloring Agents; D007202:Indicators and Reagents; D012394:Rosaniline Dyes; C004692:coomassie Brilliant Blue