Improved Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis of Urinary Proteins for Assessing Renal Function: Method Development and Preliminary Assessment

Abstract

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) analysis of urinary proteins has been employed by many investigators to assess the renal toxicity of various xenobiotics. The present study was designed to evaluate the resolution, reproducibility, and sensitivity of an improved method for this technique. Daiichi MiniPLUS precast SDS gels (10–20% gradient) were used for all tests. The gels were electrophoresed in a Daiichi 2-GEL or 6-GEL Device at an input voltage of 170 V, a starting current of 30 mA per gel, and a power ceiling of 8 W for ∼2.5 h. A double staining method of ProBlue followed by Daiichi Silver Stain II was employed and the protein bands were quantified using laser densitometry. Intra- and intergel reproducibility of electrophoretic mobility was 1.77 and 1.48% (CV), respectively, for proteins ranging from 14 to 116 kDa. Double staining allowed for the detection of as little as 20 ng of protein. To demonstrate the sensitivity and precision of this method 12-h pooled urine samples were collected and analyzed sequentially over a period of 3 days from normal men and women between the ages of 18 and 45. The study revealed detailed information about the changes in the proteinuria profiles with regard to sex, age, and the diurnal cycle. The increased sensitivity, reproducibility, and speed associated with this method enhances the potential of SDS-PAGE as a practical, non-invasive method for evaluating nephrotoxic compounds and monitoring changes in renal function.

Key Words: :

• SDS-PAGE
• Nephropathy
• Proteinuria