solution of bovine serum albumin(BSA)(see Note 4). 3.2 Microassay 1.To 1.0mL of aqueous protein solution containing 0.5-1.0 ug of protein/mL add 1 mL of SWR. 2.Incubate at 60 C for 1 h. 3.Cool,and read the absorbance at 562 nm. Notes 1.Reagents A and Bare stable indefinitely at room temperature.They may be purchased ready prepared from Pierce,Rockford,IL. 2.The sensitivity of the assay can be increased by incubating the samples longer.Altematively,if the color is becoming too dark.heating can be stopped earlier.Take care to treat standard on e the olor dovdlost ble tt I 4.Note.that like the Lowry assay.response to the BCA assay is dependent on the amino acid composition of the protein.and therefore an absolute concentration of protein cannot be determined.The BSA standard curve can only therefore be used to compare the relative protein coneentrationofsinmilarproteinsolutions. s.Some re sinterfere with the BCA assay,but nothing like asm any as with the Lowry assay (see Table 1).The presence of lipids gives excessively high absorbances with this assay. Variations produced by buffers with sulfhydryl agents and detergents have been deseribed. 6.Since the method relies on the use of Cu,the presence of chelating agents such as EDTA will of course severely interfere with the method.However.it may be possible to overcome such mains high to the assay (see Table 1).In each case it is of course necesary to run an appropriate control sample to allow for any residual color development.A modification of the assay has been deseribed that overcomes lipid interference when measuring lipoprotein protein content. Table 1 Effect of Selected Potential Interfering Compounds" BCA assay (pg BSA found) Lowry assay (ug BSA found) Sample (50 gg BSA) Water blank Interference blank Water blank Interference blank in the following corrected 0.00 420 43 0.IN NaOH 49.00 50.60 5060 0.2%Sodium azide 51.10 50.90 4920 49.00 002%Sodium azide 51.10 51.00 49.50 49.60 1.0 M Sodium由Lorid 51.30 51.10 50.20 50.10 100 mM EDTA(4 Na 50 mM EDTA (4 Na 20 29.40 10 mM EDTA (4 Na) 48.80 49.10 33.60 12.70 50 mM EDTA (4 Na).pH 11.25 31.50 32.80 T2.30 5.00