Total Protein Concentration Measurement Protocol

The Biuret Reaction:

    This protein assay is a dye-binding assay based on the observation that the absorbance maximum for an acidic solution of coomassie brilliant blue (G-250) shifts from 465 nm to 595 nm when binding to protein occurs. Therefore, we take advantage of this as a method to measure protein concentrations.

1. First we need to know how known concentrations of a protein will interact with the dye reagent and what the resulting absorbance values will be. We can then use this information to construct a standard curve that will us to determine unknown protein concentrations from their absorbance values using the same dye reagent.
 

a) Label eight small  test tubes 1 through 8 and add BSA (bovine serum albumin) and water in the amounts indicated below. Gently mix the BSA and water by gently mixing each tube.
 
 

                                    1         2              3              4                5              6                  7                8

BSA 0.25 mg/ml          -         0.1            0.2           0.4             -               -                 -                 -

BSA 1.0 mg/ml            -          -               -                -               0.2         0.3             0.4             0.5

Distilled water             0.5       0.4            0.3           0.1             0.3         0.2             0.1             -
 

b) Add 100 microliters of each tube of BSA standard (1-8 from above) to an appropriately labeled large tube.
c) Add 5 ml of BioRad dye reagent to each tube - mix.
d) Let stand at room temperature for 20 minutes and read the absorbance of each tube at 595 nm in a  spectrophotometer. Pour the sample into the cuvette to read in the spec. Use sample 1 as a blank. Use one cuvette for all readings, starting with the lowest concentration to the highest.

Construct a standard curve from this data. On the y-axis plot the absorbance value. On the x axis plot the concentration of the BSA.

To measure the protein concentrations of the fractions collected from the dialysate run through the column and the fractions collected from the control column, add 100 microliters of the lysate to 5 ml of dye reagent, wait 20 minutes, and read the OD 595 as you did for the BSA samples. Determine to amount of total protein in mg/ml in the fractions by using the standard curve.
 

                                                  Determining Specific Activity and Enzyme Units

Specific Activity

1.    Determine the lysozyme activity per minute from your lysozyme assay for all fractions tested. (Select two
       absorbance unit changes over one minute and average them). Since you only added 500 microliters of each fraction,
        normalize this number to 1 ml by multiplying it by 2.

2.    Divide this number by the total protein concentration in that same time point (measured in mg/ml), determined from
       the Biuret assay. This value gives you lysozyme specific activity relative to the total original culture. Remember, we
       began with 40 ml of culture. Divide this number by 40 to get the specific activity per ml of culture. Omit this
       extra step for the control column fractions that used purified material. Record these results to include in your
        lab report as a separate table. From this data, can you determine what salt concentration elutes the lysozyme from
       the columns?

Calculating Enzyme Units

1    The official definition of an Enzyme Unit (EU) is the amount of enzyme that causes a change in OD450 of
       0.001 absorbance units per minute (pH 7.0, 25oC).

2    Calculate the total number of EUs in each fraction of both columns. For the dialysate
       fractions, once again, normalize the number to 1 ml of culture by dividing by 40. Report all data in your lab report in
       a separate table. Again, can you determine from this data where the lysoyme elutes from the columns? Does this
       correlate with the specific activity data?