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Routine diagnosis with PhastSystem compared to conventional electrophoresis: Automated sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, silver staining and Western blotting of urinary proteins
Author(s) -
Scherberich Jürgen E.,
Fischer Peter,
Bigalke Annette,
Stangl Pia,
Wolf Gunter B.,
Haimerl Marianne,
Schoeppe Wilhelm
Publication year - 1989
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150100114
Subject(s) - silver stain , chromatography , polyacrylamide gel electrophoresis , gel electrophoresis of proteins , gel electrophoresis , sodium dodecyl sulfate , staining , chemistry , blot , polyacrylamide , electrophoresis , albumin , microbiology and biotechnology , biochemistry , biology , enzyme , genetics , gene
The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient‐gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis (SDS‐PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional “macro”‐method with self‐cast SDS‐polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 μL sample volumes and an 8–25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS‐PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha‐1‐antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS‐PAGE and staining allows easy standarization of urine SDS‐PAGE among clinical routine laboratories.