Bradford protein assay and the transition from an insoluble to a soluble dye complex: effects of sodium dodecyl sulphate and other additives.
The addition of sodium dodecyl sulphate (SDS) (0.0015-0.006%), phenol (0.25-0.5%) or sodium hydroxide (0.025-0.1 M) to the Bradford dye reagent does not improve the solubility of the Coomassie blue-protein dye complex. Centrifugation of the assay tubes, 10 min after the addition of reagent, results in complete loss of colour yield as indicated by the absorbance (A595) of the recovered supernates. At protein-concentrations above the working range of the assay, centrifugation indicates a transition from an insoluble to a soluble protein-dye complex. This transition is characteristic of an individual protein and is influenced by assay modification. Low protein concentrations appear to provide nucleation sites for precipitation of Coomassie blue whilst higher protein concentrations increase its solvation. A soluble dye chromophore is only formed above the working range of the assay indicating that precipitation of the dye by protein contributes to the assay mechanism.
Marshall
T
T
School of Health Sciences, University of Sunderland, UK.
Williams
K M
KM
D002498:Centrifugation; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012394:Rosaniline Dyes / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D012995:Solubility
D011506:Proteins; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; C004692:coomassie Brilliant Blue