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 2 dialysatess run through the columns lat week and the fractions collected from the control column, add 100 microliters of each 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 Enzyme Units

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 lysozyme columns.

3.   Calculate the total number of EUs in the original undiluted stock lysozyme.

4.   Calculate the total number of EUs in the protein from the expressed lysozyme culture.