Clinical analysis of human urinary proteins using high resolution electrophoretic methods

Abstract

The application of isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis (2-DE) and capillary electrophoresis (CE) for high resolution electrophoretic analysis of human urinary proteins is reviewed. In each case, the information is tabulated chronologically with details of sample preparation, electrophoretic system, detection method and clinical application. The text includes an historical perspective of the use of each method for urinalysis and a detailed review of the application of the methods to the investigation of renal disease, renal transplantation, Bence Jones proteinuria (BJP), diabetes mellitus, cadmium toxicity, nephrolithiasis and cancers of the urogenital tract.

Isoelectric Focusing Sample Injection for Capillary Zone Electrophoresis in a Fused Silica Capillary

“…1,2 However, conventional CE is limited to a relatively poor concentration detection limit due to the low volume loadability of the capillary; 3,4 also, sample preconcentration is always considered before CE separation. [5][6][7][8][9][10][11][12][13][14][15] Among various concentration methods, isoelectric focusing (IEF) has been demonstrated to be an effective approach for the concentration of proteins. 14,15 In a conventional IEF, carrier ampholytes are used to establish a stable pH gradient.…”

“…[5][6][7][8][9][10][11][12][13][14][15] Among various concentration methods, isoelectric focusing (IEF) has been demonstrated to be an effective approach for the concentration of proteins. 14,15 In a conventional IEF, carrier ampholytes are used to establish a stable pH gradient. 16 Recently, IEF without carrier ampholytes 17 and carrier ampholyte free-IEF 18 are also being developed, and have shown the feature of a high concentration factor.…”

Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain

“…Numerous colorimetric methods for staining proteins and peptides in isoelectric focusing gels have been devised over the years, including Coomassie Brilliant Blue (CBB R-250, CBB G-250), colloidal CBB, CBB/Crocein Scarlet, Fast Green, colloidal Acid Violet 17, and silver staining [18±24]. CBB and silver staining are most routinely utilized for protein visualization in both IEF and IPG electrophoresis [5,25,26]. Fluorescent detection of proteins in IEF gels has also been demonstrated using Nile red [27].…”

Electrophoretic analysis of Bence Jones proteinuria

“…Electrophoretic methods have been widely used for the clinical analysis of proteinuria (protein in the urine) [1]. Under normal circumstances, the renal glomeruli restrict filtration of plasma proteins and the renal tubules reabsorb and degrade most filtered protein such that only trace amounts of protein (approximately 150 mg per 24 h) appear in the urine [2].…”

“…In renal disease, the level of protein in the urine tends to be persistently elevated and the protein composition is modified in a characteristic manner. Renal glomerular disease (glomerulonephritis, nephrosis, glomerulosclerosis and IgA nephropathy) results in urine containing increased amounts of albumin and high molecular weight proteins whilst renal tubular disease (pyelonephritis, drug-induced tubulopathies and heavy metal toxicity) results in urine containing elevated levels of low molecular weight proteins such as b 2 -microglobulin, lysozyme, retinol-binding protein, a 1 -microglobulin, and a 1 -acid glycoprotein [1,2]. Alternatively, excess levels of abnormal protein in the blood can spill over into the glomerular filtrate ± this overloads the reabsorption capacity of the renal tubules, resulting in the excretion of the excess protein in the urine [2].…”