The solubility of Coomassie blue protein-dye complexes.

Coomassie blue protein dye-binding assays are commonly used for quantitation of protein. They exploit the interaction between protein and Coomassie brilliant blue (CBB) G-250, the latter being solubilized in either ethanol/phosphoric acid (1) or perchloric acid (2). The Bradford assay (1) is the method of choice. Potential problems with these assays include non-linearity, a variable response to different proteins and a tendency for the protein-dye complex to precipitate on prolonged standing (1-5). However, it is generally believed that the assay measures formation of a soluble blue complex providing readings are taken between 10 and 60 min after addition of the reagent. We have recently shown that centrifugation of assay tubes (10 min after reagent addition) results in loss of colour yield (with a range of proteins) irrespective of whether the dye reagent contains ethanol/ phosphoric acid or perchloric acid (6,7). Thus, the initial protein-dye complex is insoluble at the time of absorbance measurement. Colorimetric assays shoul measure formation of a soluble chromophore. We have sought to achieve this with the Bradford assay using different assay modifications. It is also important to recover microgram amounts of protein from dilute solution and we have exploited CBB precipitation for this purpose. Further work, at higher protein amounts, indicates a transition from an insoluble to a soluble protein-dye complex. This is reproducible for an individual protein but varies from protein to protein. Protein solutions were prepared gravimetrically (15 mg/mL) in 0.15 mol/L sodium chloride or ultrapure water (MilliQ) and centrifuged (12,000 rpm, 5 min;MSE Micro Centaur Centrifuge) prior to use. Dye reagent (0.1 g Serva Blue G in 50 mL of 95% ethanol, 100 mL of 85% w/v phosphoric acid, 850 mL water) wasfiltered immediately prior to u s e . For assay, 1 mL of dye reagent was added to 1.5-200 ug of protein in microfuge tubes and the absorbance (595 nm) measured against a reagent blank. The assay tubes were centrifuged (as above) and the A595 of the supernates measured against a centrifuged reagent blank. In subsequent experiments the composition of the dye reagent was modified and precipitation of protein confirmed by washing the dye pellets in acetone and resolubilizing in SDS sample denaturing solution prior to SDS-PAGE (8). Protein bands were detected by silver staining (9) and recovery monitored by densitometry. Modification of the dye reagent by addition of SDS (0.003%) (lo), Triton X-100 (0.008%) (11). phenol (0.5%) (12) or sodium hydroxide (0.05 M) (13) still gave an insoluble complex. Reducing the dye concentration from 0.01% to 0.005% (4) gave a solublecomplex with albumin but negligible colour yield with other proteins. Lowering the phosphoric acid reversed this effect. The results indicate that a saturating concentration of dye and the formation of an insoluble protein-dye complex are essential pre-requisites for good colour yield and uniform protein response. The protein appears to provide nucleation sites for precipitation of the dye.