Concentrations of albumin in the final assay volume (f.a.v.) of 5 ml may range from ~30 to 300 jug protein/ 5 ml f.a.v. to produce A650 readings from ~0.20 to 0.80.

2. Add 1.0 ml of the protein-containing sample, of each dilution of calibration standard, or of the buffer or solvent used to prepare the sample (reference standard) to 0.90 ml of Hartree-Lowry reagent A in separate test tubes. Incubate 10 min in a 50°C water bath.

3. Cool the tubes to room temperature.

4. Add 0.1 ml of Hartree-Lowry reagent B to each tube and mix. Incubate 10 min at room temperature.

5. Rapidly add 3 ml Hartree-Lowry reagent C to each tube and mix thoroughly. Incubate 10 min in a 50°C water bath, then cool to room temperature.

The final assay volume is 5.0 ml.

6. Measure the net absorbance the sample, calibration standards, and reference standard at 650 nm (A650) in 1-cm cuvettes. If the spectrophotometer does not automatically give net absorbance readings, subtract the value for the reference solution from those obtained for the sample and calibration standards.

7. Prepare a calibration plot by graphing the net A650 values for the standards versus protein concentration (^g protein/5 ml f.a.v.). Determine the protein concentration of the sample by interpolation from the plot.

Plot the data as shown in Figure 3.4.2 and calculate the slope and its units. The slope of a calibration plot is proportional to the spectrophotometric absorption coefficient, and is a measure of the sensitivity of assay over the range indicated on the horizontal axis. Comparisons can be made with other measures, such as the weight absorption

Bovine Serum Albumin Absorbance

Figure 3.4.2 Hartree-Lowry assay calibration plot using bovine serum albumin. Slope = 2.5 x 10 A650 (|ig protein/ml f.a.v.)-1 cm-1.

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