Ultrathin‐layer sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis and silver staining of urinary proteins

Abstract

Ultrathin-layer sodium dodecyl. sulfate-polyacFylamide gradient gel electrophoresis in combination with silver staining is a highly sensitive and rapid method for molecular size analysis of urinary proteins. First results are achieved after 4 h (electrophoretic run, 100 min, followed by Coomassie Brilliant Blue staining, 2 h). After additional silver staining, including a recycling step, the results are ready for evaluation within 7 h. Urine samples do not need to be concentrated. Within a single gel, 25-27 samples can be run side by side under identical conditions and thus compared without ambiguity. By coelectrophoresis of pure proteins and immunological analysis 13 urinary proteins could be identified and for 7 of them the detection limits were determined. Glomerular protein patterns in samples of urine with a protein concentration in the normal range are compared with the concentration of albumin and transferrin measured by a sensitive enzyme immunoassay. Typical renal and extrarenal protein patterns are shown.

Clinical analysis of human urinary proteins using high resolution electrophoretic methods

“…Whilst retinol binding protein and P,-microglobulin were sufficiently resolved to allow detection by CBB staining, silver staining revealed many LMW proteins and this complicated the classification of tubular proteinuria [81]. Schiwara et al [82] and Shiba et al [83], in similar studies, combined SDS-PAGE, silver staining and immunological analysis to detectlidentify 26 urinary proteins (M, 14 000-230 000) [83] and determine the detection limits of seven [82]. The a1bumin:transferrin ratio (for differentiation of glomerular proteinuria) was determined by SDS-PAGE or enzyme immunoassay and direct comparison showed good correlation between the two methods [82].…”

“…Schiwara et al [82] and Shiba et al [83], in similar studies, combined SDS-PAGE, silver staining and immunological analysis to detectlidentify 26 urinary proteins (M, 14 000-230 000) [83] and determine the detection limits of seven [82]. The a1bumin:transferrin ratio (for differentiation of glomerular proteinuria) was determined by SDS-PAGE or enzyme immunoassay and direct comparison showed good correlation between the two methods [82]. Tamm Horsfall mucoprotein was not detected unless the sample was treated with SDS [83].…”

Renal tubular dysfunction in patients with inflammatory bowel disease treated with aminosalicylate.

“…An aliquot was taken and frozen for determination of enzyme activity. The morning urine was subjected to protein analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as described by Pesce et a12' and Schiwara et al 22 The analysis of microproteinuria by SDS-PAGE allows the differentiation between glomerular and tubular proteinuria by protein size and pattern. The detection of significant amounts of protein with a tubular pattern, which represents proteins originating from the tubular epithelium, was considered a pathological finding.2' 22 The lower sensitivity limit for urinary protein detection and analysis by SDS-PAGE was 5 mg/l, the reference range was between 7 and 56 mg/24 h (90% confidence interval).…”

Sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns of proteinuria in various renal diseases of childhood

“…These include: the use of thin gels, silver staining that is more sensitive than Coomassie blue [5], and Western blotting methods for the identification of individual protein bands on the electrophorograms [6,7]. We have recently examined the potential of the PhastSystem, an automated electrophoretic system produced by Pharmacia (Uppsala, Sweden), that can produce ready-to-read SDS PAGE separations of 1 gl samples of unconcentrated urine in 2 h [8].…”