BIOL 1406

PreLab 8b.1

How can I determine the protein concentration of the 8 milk fractions that I collected during the purification of α-lactalbumin?


Last week, you used several techniques in an attempt to separate α-lactalbumin from the other molecules found in nonfat milk. At several points during the procedure, you saved samples of the milk fractions. These fractions will be analyzed using SDS-PAGE electrophoresis in order to assess the effectiveness of the various purification steps. Electrophoresis will separate the various proteins present in each milk fraction, allowing you to determine the number of proteins present and their molecular weights. Knowledge of molecular weights can then be used to help identify the various proteins present in each fraction.

Before you run gel electrophoresis, however, you must decide how much of each sample should be loaded onto the electrophoresis gel. This is important in order to avoid loading too much or too little protein onto the gel. The amount of sample needed will depend on the protein concentration of each sample. Therefore, your job today will be to estimate the total protein concentration of each milk fraction that you collected last week.

A protein assay is a test that is used to detect the presence of protein. A qualitative assay simply indicates whether protein is present in your sample. Some qualitative assays will detect any protein, while others are designed to detect a specific protein. A quantitative assay, on the other hand, is used to determine how much protein is present. A quantitative assay may be used to measure the concentration of protein in a solution (e.g. in mg/mL), or, in the case of an enzyme, the assay may be designed to measure the level of enzyme activity. During protein purification, it is useful to have a quantitative assay to determine how much protein is present after each step of the purification process. This allows scientists to monitor the concentration and/or activity of the protein as it is being purified.

In today’s lab, you will determine the concentration of protein in each of the 8 milk fractions that you prepared during Lab 8A:

• Skim milk
• Pellet
• Supernatant
• The 5 column fractions obtained from size exclusion chromatography of the whey proteins

There are several methods for determining the concentration of protein in a solution. One method, which was mentioned in Prelab 8a.3, is absorbance of light at 280 nm (A280). This assay has the advantage of being quick and easy; you simply place the sample in a spectrophotometer and read the absorbance. In addition, this method is nondestructive, so it does not use up any of the sample that is being analyzed. Unfortunately, this assay is not very accurate because it depends on the concentration of amino acids with rings in their R-groups, and different proteins have different amounts of those amino acids.

The biuret and Lowry assays are both based on the binding of copper ions (Cu2+) to the nitrogen atoms present at the peptide bonds of proteins. This reaction causes the protein solution to turn purple (biuret) or blue (Lowry). The color intensity is proportional to the concentration of protein in the sample. The color intensity is measured using a spectrophotometer, and the results are compared to a standard curve prepared using known concentrations of a purified protein standard. Each of these assays has advantages and disadvantages. The biuret assay is the most reliable assay for protein because other substances present in the solution rarely interfere with the accuracy of the results. Unfortunately, it takes about 20-30 minutes for the color to develop, so it isn’t nearly as fast as the A280 assay, and it can only detect relatively high concentrations of protein (1 mg/mL or more.) The Lowry assay, in which a second reagent is added to the biuret reagent, is much more sensitive and can be used to measure protein concentrations as low as 5 μg/mL. However, the Lowry assay takes even longer to complete, requiring 40-60 minutes for the color to develop. Reliability is another disadvantage of the Lowry assay because other substances in the solution can greatly affect its accuracy.

BSA samples used to prepare a standard curve for the Bradford's assay

The Bradford assay is the most commonly used protein assay because of its accuracy, sensitivity, and convenience. The Bradford assay is much more accurate than A280 measurements, few other substances interfere with its accuracy, it is sensitive to protein concentrations as low as 1 μg/mL, and it is very rapid, producing a color change within five minutes. The Bradford assay is based on the binding of Coomassie Blue dye to proteins. The dye alone has a brownish color and absorbs light most efficiently at 465 nm. When the dye combines with protein, however, the resulting protein-dye complex has an intense blue color whose maximum absorption is at 595 nm.


In today’s lab, you will use the Bradford assay to determine the protein concentration of the 8 milk fractions you saved last week. In order to determine the protein concentration of these milk fractions, you will need to prepare a standard curve that shows the exact relationship between the A595 value of each fraction and its protein concentration.

To prepare a standard curve for the Bradford assay, first prepare a series of solutions of known protein concentration using bovine serum albumin (BSA), a protein commonly used as a protein standard. Next, add Bradford’s Reagent (containing Coomassie Blue dye) to the BSA solutions and measure the A595 value of each solution. Next, prepare a scatter diagram of your data by plotting protein concentration of the BSA solutions on the x-axis and A595 values on the y-axis. Now, use linear regression to determine the equation of the best-fit straight line for your data points. This equation is the equation of your standard curve; it shows the exact relationship between protein concentration and the A595 value of a solution.

Once you have the linear regression equation for your standard curve, you can use the Bradford assay to determine the protein concentrations of the 8 milk fractions you prepared last week. First, dilute the 8 milk fractions so their A595values will lie within the range of your standard curve. Next, add Bradford’s Reagent to each diluted milk fraction and measure its A595 value. Now, substitute these A595values into your linear regression equation for “y” and calculate the corresponding values for “x”. The “x” values equal the protein concentrations of the diluted milk fractions. Finally, calculate the protein concentrations of the undiluted milk fractions.
 

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