Determining total protein content of urine: centrifugation results in precipitation of the protein-coomassie brilliant blue dye complex.

The Coomassie Brilliant Blue (CBB) protein dye-binding assay (1) has been favorably assessed for determining the total protein content of urine (2-4), although discrepancies have been noted in its response to different proteins (5) and sample dilution (6). It is commonly accepted that the proteinCBB dye complex is soluble under assay conditions, but our studies reveal loss of color yield after microfugation or filtration of assay tubes containing protein standard or human urine. Procipitation ofthe complex occurs immediately after dye reagent is added and could cause problems in automated analysis. Three urine pools, each comprising 8-10 specimens, were prepared by mixing equal volumes of early morning midstream samples. Bovine albumm (protein standard) was freshly preparodat1g/L(basedon1%A= 6.6) in 0.15 molIL sodium chloride and microfuged immediately before assay. The CBB dye reagents were prepared as recommended (1, 7, 8) or purchased from Bio-Rad Labs. (Richmond, CA). The increase in absorbance (A595 or Aem) was measured against a reagent blank after 10 mm with a Jenway Model 6100 spectrophotometer (Dunmow, UK). The assay mixtures were then microfuged (13 400 x g, 5 mm; M.S.E. Micro Centaur Centrifuge, Loughborough, UK) or filtered (0.2 pm; Minsart ifiter unit, Sartorius, Belmont, UK) and absorbance measurements were repeated on the respective supernatants and filtrates. The assay protocols were as follows: . Bradford microassay (1 ): We added 1 mL of Bradford dye reagent [per liter: 0.1 g CBB G-250 (Sigma Chemical Co., St. Loins, MO), 1.6 mol phosphoric acid, and 0.8 mol ethanol] to 5-20 ig samples of the standard protein (0.2 gIL) or 25-100 L ofthe urine pool in a sample volume of 0.1 mL. . Read and Northcote standard assay (7): We added 950 iL of dye reagent [per liter: 0.1 g Blue G (Serva, Heidelberg, FRG), 1.6 mol phosphoric acid, and 0.8 mol ethanol) to 5-20 .ig samples of the standard protein (0.4 g/L) or 5-50 pL of the urine pool in a sample volume of 50 L. . Bio-Rad microassay: We added 0.2 mL of the commercial dye reagent concentrate to 2.5-10 g samples of the standard protein (0.015 g/L) or 100-400 LL of the urine pool in a sample volume of 0.8 mL. . Sedmak and Grossberg standard assay (8): We added 500 iL of dye reagent (per liter: 0.6 g Serva Blue G and 0.3 mol perchloric acid) to 25-100 2g of standard protein (1 g/L) or 100500 pL of the urine pool in a sample volume of 500 L. Figure 1 shows the color yield respouse of the Read and Northcote standard assay to human urine and protein standard. Microfugation resuited in visible precipitation of the blue protein-CBB dye complex with consequent loss of color yield in the respective supernatanta (Figure 1). Similar results were obtained with the Bradford microassay and the Sedmak and Grossberg standard assay. In contract, the Bio-Rad microassay showed incomplete precipitation with an average of 60% recovery in the urine supernates and 30-90% recovery in the protein-standard supernatants (recovery increased with standard protein