This exercise introduces students to four methods of determining protein concentrations: absorbance at 280 nm, and the biuret, Lowry, and Bradford assays. (I haven't yet incorporated the BCA assay; I'm trying to balance thoroughness with available time.) The goals of the exercise are to let students see and evaluate these commonly-used assay methods. I use just two proteins, bovine serum albumin (BSA) and gelatin. BSA is fairly expensive, but 100 g (over $100) lasts many years. Egg albumin would work, probably, but it seems if I just stare at it too long, it coagulates, whereas BSA forms a clear, stable solution. I prepare solutions of BSA and gelatin at 0.1, 0.2, 0.3, 2, 4, and 6 mg/mL in water. The latter three are for the biuret assay and the former are for the rest. The assay procedures are as follows:

Biuret: Mix 0.50 mL of protein with 2.50 mL of biuret reagent and measure the absorbance at 540 nm.

A₂₈₀: Measure the absorbance of 1 mL at 280 nm.

Lowry: Mix 0.25 mL of protein with 2.5 mL of Lowry reagent 1. After 10 minutes, add 0.25 mL of Lowry reagent 2 and mix well immediately. After 30 minutes, measure the absorbance at 750 nm (if you're using a Spectronic 20 with a normal phototube, 750 may be too long; 600 nm gives lower absorbances but works okay).

Bradford: Mix 0.25 mL of protein with 2.5 mL of Bradford reagent and measure the absorbance at 595 nm.

All series should include a zero protein (water) tube (reagent blank). I have the students zero their instruments to the reagent blanks. This is especially important with the Bradford assay, for which the reagent blank has quite a high absorbance (>0.5). Two other practical concerns with the Bradford assay are its great sensitivity to detergent (tubes must be rinsed very well) and the fact that it stains the cuvettes blue. I don't know whether this is a problem during data collection, but is it easily washed out.

After plotting their data on separate graphs for each assay, students are asked to evaluate each method by the criteria of convenience (how easy is it to do?), sensitivity (how well does it detect small amounts of protein; for example, what mass of protein is needed to give an absorbance of 0.1?), generality (how consistent are the results among the two different proteins?), and linearity (does it give a straight line plot of absorbance vs. protein?).

Revised 6/11/07