Lowry Protein Assay

LOWRY PROTEIN ASSAY The Lowry protein assay has been used by biochemists since the original work was published in 1951*. 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 by the equation shown below. TOTAL VOLUME 1.00 mL 800 mL 1st reagent / 10 minute incubation 100 mL 2nd reagent / 1.5 hours at RT DATA FOR STANDARD PLOT µ L BSA µ L H2O µ g BSA A660 1/µ µ g BSA A660 1 CALC A660 corr 0.400 1/A660 1/A660 CALC2 14.93 7.14 4.78 3.70 3.01 2.54 2.23 1.92 1.74 1.61 14.04 7.13 4.83 3.68 2.99 2.53 2.20 1.95 1.76 1.61 0.23 mg/mL 0 10 20 30 40 50 60 70 80 90 100 100 90 80 79 60 50 40 30 20 10 0 0 4 8 12 16 20 24 28 32 36 40 0.032 0.099 0.172 0.241 0.302 0.364 0.426 0.480 0.553 0.606 0.655 0.000 0.067 0.140 0.209 0.270 0.332 0.394 0.448 0.521 0.574 0.623 0.000 0.071 0.140 0.207 0.272 0.335 0.396 0.455 0.512 0.568 0.622 0.250 0.125 0.083 0.063 0.050 0.042 0.036 0.031 0.028 0.025 0.000 1 A660 = µg BSA / (m + b * µg BSA ) 2 1 / A661 = m / µg BSA + b For double reciprocal plot: SUMMARY OUTPUT DATA AND ANALYSIS FOR SAMPLES Regression Statistics Mult R 0.99989 R Square 0.99977 Adj R Sq 0.99974 Std Error 0.02921 Observ 9 Coef Std Error b 0.22555 0.0195 m 55.2632 0.3164 UNK = A660 * m / (1 - A * b ) CONTROL µL A660 10 10 20 20 0.164 0.153 0.247 0.243 A660 corr UNK µg µ g/µ µL 0.132 0.121 0.215 0.211 7.52 6.87 12.5 12.2 0.752 0.687 0.624 0.612 A660 corr UNK µg µ g/µ µL 0.121 0.119 0.218 0.237 6.87 6.76 12.7 13.8 0.687 0.676 0.634 0.692 SUMMARY TREATED µL A660 10 10 20 20 0.153 0.151 0.250 0.269 AVE µ g/µ µL CONTROL TREATED 0.669 0.672 % 100 100 0.800 0.700 ABSORBANCE 0.600 0.500 0.400 0.300 0.200 0.100 0.000 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 µg BSA Figure 1a. Hyperbolic behavior of Lowry protein assay. The line is a fit to the slope (m) and y-intercept (b) of the double reciprocal plot in Figure 1b. 16.00 14.00 INVERSE ABSORBANCE 12.00 10.00 8.00 6.00 4.00 2.00 0.00 0.000 0.050 0.100 0.150 0.200 0.250 INVERSE µg BSA Figure 1b. Double reciprocal plot for the Lowry protein assay. The line is calculated from the slope (m) and y-intercept (b) from a regression analysis of the data.