Bio-RadProtein AssayFor Technical ServiceCall Your Local Bio-Rad Office orin the U.S. Call 1-800-4BIORAD(1-800-424-6723)LIT33C 8/31/98 02:25 PM Page aTable of ContentsSection 1 Introduction..................................................................... 11.1 Principle ..................................................................................... 11.2 Product Description.................................................................... 31.3 Materials Required but Not Supplied......................................... 3Section 2 Instructions...................................................................... 42.1 Reconstituting the Standard ....................................................... 42.2 Standard Procedure .................................................................... 42.3 Microassay Procedure ................................................................ 52.4 Microtiter Plate Protocols .......................................................... 6Section 3 Common Questions......................................................... 8Section 4 Troubleshooting Guide ................................................... 12Section 5 Ordering Information..................................................... 13Section 6 Safety Information .......................................................... 14Section 7 References........................................................................ 24LIT33C 8/31/98 02:25 PM Page b1Section 1IntroductionThe Bio-Rad Protein Assay, based on the method of Bradford, is asimple and accurate procedure for determining concentration of solubi-lized protein. It involves the addition of an acidic dye to protein solution,and subsequent measurement at 595 nm with a spectrophotometer ormicroplate reader. Comparison to a standard curve provides a relativemeasurement of protein concentration. 1.1 PrincipleThe Bio-Rad Protein Assay is a dye-binding assay in which a differ-ential color change of a dye occurs in response to various concentrationsof protein.1The absorbance maximum for an acidic solution ofCoomassie®Brilliant Blue G-250 dye shifts from 465 nm to 595 nm whenbinding to protein occurs.2,3,4 The Coomassie blue dye binds to primarilybasic and aromatic amino acid residues, especially arginine.5Spector6found that the extinction coefficient of a dye-albumin complex solutionwas constant over a 10-fold concentration range. Thus, Beer’s law may beapplied for accurate quantitation of protein by selecting an appropriateratio of dye volume to sample concentration. Interferences may be caused by chemical-protein and/or chemical-dyeinteractions. Table 1 lists those chemical reagents not directly affectingthe development of dye color. (Note: Basic buffer conditions and deter-gents interfere with this assay.) Since every protein-chemical reagentcombination has not been assayed, it is possible that some of the listedreagents produce interference in combination with certain proteins.However, with respect to proteins such as bovine serum albumin andgamma globulin, the listed reagents show little or no interference. Theacceptable concentrations of reagents for the Standard Procedure areshown in Table 1. Equivalent concentrations of reagents for theMicroassay Procedure (see Section 2) are 1/40 of those listed in this table,due to the difference of sample-to-dye ratios between the Standard andMicroassay Procedures. LIT33C 8/31/98 02:25 PM Page 12Table 1. Reagents Compatible with the Bio-Rad Protein AssayWhen Using the Standard Procedure.*Acetate, 0.6 M K CI, 1.0 MAcetone Malic a cid, 0.2 MAdenosine, 1 mM Mg Cl2, 1.0 MAmino Acids Mercaptoethanol, 1.0 MAmmonium sulfate, 1.0 M MES, 0.7 MAmpholytes, 0.5% MethanolAcid p H MOPS, 0.2 MATP, 1 mM Na C l, 5 MBarbital NAD, 1 mMBES, 2.5 M NaSC N, 3 MBoric acid PeptonesC a codylate-Tris, 0.1 M Phenol, 5%C DTA, 0.05 M Phosphate, 1.0 MCitrate, 0.05 M PIPES, 0.5 MDeoxycholate, 0.1% Polyadenylic a cid, 1 mMDithiothreitol, 1 M Polypeptides (MW<3000)DNA, 1 mg/ml Pyrophosphate, 0.2 MEDTA, 0.1 M rRNA, 0.25 mg/mlE G TA, 0.05 M tRN A, 0.4 mg/mlEthanol total RNA, 0.30 mg/mlEagle’ s MEM SDS, 0.1%Earle’ s salt solution Sodium phosphateFormic acid, 1.0 M Stre ptomycin sulfate, 20%Fructose Triton X-100, 0.1%Glucose TricineGlutathione Tyrosine, 1 mMGlycerol, 99% Thymidine, 1 mMGlycine, 0.1 M Tris, 2.0 MGuani dine-H CI Urea, 6 MHank's salt solution VitaminsHEPES buffer, 0.1 M* Interference may be caused by chemical-protein and/or chemical-dye interactions. Table 1 liststhose chemical reagents not directly affecting the development of dye color. Since every protein-chemical reagent combination has not been assayed, it is possible that some of the listed reagentsproduce interference in combination with certain proteins. However, with respect to proteins such asbovine albumin and globulin, the above listed reagents show little or no interference.LIT33C 8/31/98 02:25 PM Page 231.2 Product DescriptionProtein Assay Dye Reagent Concentrate (catalog number 500-0006)contains 450 ml of solution containing dye, phosphoric acid, andmethanol. One bottle of dye reagent concentrate is sufficient for 450assays using the standard assay procedure, or 2,250 assays using themicroassay procedure.The Dye Reagent Concentrate can be purchased in a kit with one oftwo standards: Bovine gamma globulin (Kit I, catalog number 500-0001)or bovine serum albumin (Kit II, catalog number 500-0002).The Bio-Rad Protein Assay is for research use only.1.3 Materials Required but Not SuppliedFor standard assaySpectrophotometer set to 595 nmCuvet tes with 1 cm path length matched to laboratory spe ctropho-tometer. Bio-Ra d’ s disposable polystyrene cuvettes (catalog numb er223-9950) are recommend e d13 x 100 mm test tubesTest tube rack for 13 x 100 mm test tubesVortex mixerWhatman #1 filter (or equivalent) and funnel for dye re a g ent prepara-tionGraduate d cylinders, pipets, and containers for rea g ent pre p arationand st oragePipets a c curately d elivering 100 µl and 5.0 mlFor microplate assayMicrotiter platesPipets a c curately d elivering 200 µl and 800 µlLIT33C 8/31/98 02:25 PM Page 34Section 2Instructions2.1 Reconstituting the StandardTo reconstitute the lyophilized bovine gamma globulin and bovineserum