Sample Bradford Assay

SAMPLE BRADFORD ASSAY The Bradford protein assay is a more recently developed protein assay that is commercially available.* When a large number of points are used to define the standard plot, the data is not linear, rather it is hyperbolic. A double reciprocal plot is constructed from the standard data and the slope (m) and y-intercept (b) of the standard plot are used to determine the concentration of the unknown as shown by the equation below. Volume = 1000 µ L DATA FOR STANDARD CURVE TEST µ L BRAD µ L Water TUBE Dil 1:1* 0 1 2 3 4 5 6 7 8 9 10 200 200 200 200 200 200 200 200 200 200 200 SUMMARY OUTPUT 700 700 700 700 700 700 700 700 700 700 700 µ L TRIS 100 99 98 97 96 95 94 93 92 91 90 CONTROL 0 1 2 3 4 5 6 7 8 9 10 µ g STD 1.80 µ g/µ µL 0.0 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 ABS595 corr 0.448 0.576 0.669 0.770 0.869 0.948 1.004 1.083 1.157 1.222 1.249 Coef 0.000 0.128 0.222 0.322 0.421 0.500 0.556 0.635 0.710 0.774 0.802 1 Std Err 1/µ STD ABS596 1/µg CALC 1 0.000 0.117 0.223 0.321 0.410 0.493 0.569 0.640 0.706 0.768 0.826 0.556 0.278 0.185 0.139 0.111 0.093 0.079 0.069 0.062 0.056 0.000 1/ABS595 corr 7.800 4.513 3.108 2.375 2.000 1.798 1.576 1.409 1.291 1.248 1/ABS596 2 CALC 8.57 4.48 3.12 2.44 2.03 1.76 1.56 1.42 1.30 1.21 0.39 ABS595 calc = µgSOD/(m +µgSOD*b ) 2 b = 0.99848 0.99697 m = 0.99659 0.11981 units of 10 or 1/ABS595 calc = m /µgSO) + b 0.3935 0.837 14.721 3.8272 * BIORAD Cat. No. 500-0001 Branford, M. M. (1976). A rapid and sensitive method for m = µg the quantitation of microgram quantities of protein 14.721 µg utilizing the principle of protein-dye binding Anal. Biochem 72, 248-254. 3 UNK = A595 * m / (1 - A595 * b ) DATA FOR UNKNOWN PROTEIN SAMPLES µ L BRAD µ L Water µ L TRIS µ L UNK ABS595 ABS595 Dil 1:1 11 12 13 14 TREATED 15 16 17 18 PRO50 19 20 21 22 PRO100 23 24 25 26 ABS595 Inverse A595 vs inverse STD (µg-1) Regression Statistics Multiple R R Square Adj R Square Standard Error Observations µ L STD µ g UNK corr 200 200 200 200 700 700 700 700 98 98 96 96 2 2 4 4 0.637 0.654 0.812 0.804 0.189 0.206 0.364 0.356 3.01 3.31 6.25 6.10 1.51 1.65 1.56 1.52 200 200 200 200 700 700 700 700 98 98 96 96 2 2 4 4 0.604 0.627 0.776 0.772 0.157 0.180 0.329 0.324 2.46 2.84 5.56 5.47 1.23 1.42 1.39 1.37 200 200 200 200 700 700 700 700 98 98 96 96 2 2 4 4 0.638 0.641 0.799 0.802 0.191 0.193 0.351 0.354 3.03 3.08 6.00 6.06 1.52 1.54 1.50 1.51 200 200 200 200 700 700 700 700 98 98 96 96 2 2 4 4 0.615 0.661 0.801 0.808 0.168 0.213 0.353 0.360 2.64 3.42 6.04 6.18 1.32 1.71 1.51 1.55 1 µ g UNK3 dev ave dev ave µg µg 1.56 1.35 1.52 1.52 0.0557 0.0915 0.0016 0.0374 0.3337 0.1395 0.1731 0.1939 0.0462 0.0212 0.0632 0.0478 0.2413 0.1475 0.0518 0.0170 % DEV 0.05 3.0 0.0525 3.9 0.04 2.9 0.11 7.5 0.900 0.800 0.700 ABSORBANCE 0.600 0.500 0.400 0.300 0.200 0.100 0.000 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 SOD (µ µ g) Figure 1a. Hyperbolic behavior of the Bradford protein assay at 595 nm. The line is calculated from the slope (m) and yintercept determined from a double reciprocal plot of these data. 2 8.00 7.00 INVERSE ABSORBANCE 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0.000 0.200 0.400 0.600 INVERSE mg SOD Figure 1b. Double reciprocal plot of data from Figure 1a. The line is calculated from the slope (m) and y-intercept (b) determined from a linear regression analysis of the data. 3