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.