Preliminary treatment of urinary proteins improves electrophoretic analysis and immunodetection.
Analysis of urinary protein composition is an important tool in studies on renal physiology and physiopathology. Urine is, however, a complex mixture containing, besides protein, a variety of compounds such as salts, peptides, oligosaccharides, and glycosaminoglycans. Some of these compounds interfere with the electrophoretic migration of protein in sodium dodecyl sulfate-polyacrylamide gels and prevent correct analysis of the protein pattern. We describe a simple method for extracting urinary proteins that considerably improves their electrophoretic migration and subsequent immunodetection. This treatment involves ammonium sulfate fractionations (for precipitating proteins), EDTA (for inhibiting protein aggregation), and HCl hydrolysis (for removing glycosylaminoglycans). Recovery during extraction was found to be almost quantitative for total protein and three representative proteins: albumin, alpha 1-glycoprotein acid, and beta 2-microglobulin.
Verdier
J M
JM
Unité de Recherches de Physiologie et Pathologie Digestives, INSERM U315, Marseille, France.
Dussol
B
B
Dupuy
P
P
Berland
Y
Y
Dagorn
J C
JC
D000419:Albuminuria / Q000652:urine; D000645:Ammonium Sulfate; D015153:Blotting, Western / Q000592:standards*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000592:standards*; D005590:Fractional Precipitation; D006801:Humans; D006851:Hydrochloric Acid; D009961:Orosomucoid / Q000652:urine; D011507:Proteinuria / Q000652:urine*; D001613:beta 2-Microglobulin / Q000652:urine
D009961:Orosomucoid; D001613:beta 2-Microglobulin; D004492:Edetic Acid; D006851:Hydrochloric Acid; D000645:Ammonium Sulfate