CHEM 4401 Biochemistry Laboratory I
Protein Quantification using the Bradford Method
In this exercise we will use the Bradford protein assay to estimate the total amount of
protein in the potato PPO enzyme extract we obtained during our polyphenoloxidase
experiment. We will use this information to calculate the specific activity of PPO in our
crude enzyme preparation.
The Bradford assay relies upon the binding of proteins to a dye, Coomassie Blue. When
Coomassie Blue binds to a protein, the absorption maximum (λmax) of the dye shifts
from 465 nm to 595 nm, with a concomitant change in color from brown to blue. We
will estimate the concentration of protein in our crude extract by comparing their
absorbances to those obtained for a series of standard protein dilutions (generally Bovine
Serum Albumin)
Procedure
Preparation of a standard curve
1. Collect your potato enzyme extract from your instructor. Mix well. Prepare a
series of tubes as follows:
2. Mix tubes well. Incubate for 10 min. on benchtop.
3. Turn on spectrophotometer and set wavelength to 595 nm.
4. Zero your instrument using tube no. 7 as your blank.
5. Measure the absorbance of standards and your sample at 595 nm.
Results
1. Prepare a standard curve (Figure 1) by plotting the absorbance at 595 nm for your
standards on the ordinate (y-axis) and the amount of protein in the standards on
the abscissa (x-axis). Prepare a line of best fit to your data. Determine the
equation for your line (y = mx + b). Use the equation to calculate the
concentration of protein in your test tube sample.
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a.
b.
c.
d.
e.
Figure legend with appropriate description (1 pt)
Graph/ regression line (1 pt)
Axes labeled with appropriate units (1 pt)
Equation for regression line (1 pt)
Concentration reported in figure legend (1 pt).
2. Calculate the concentration of protein in your PPO enzyme extract (i.e account for
the dilution in your assay). Show your work (2 pt)
3. Determine the enzyme activity of PPO in your extract. Enzyme activity is
typically reported in units. One unit of enzyme activity is defined as the amount
of enzyme capable of converting 1 micromole (µmol) of substrate to product per
minute at 25°C. To calculate the number of units of PPO activity in our extracts
we must multiply the reaction rate (velocity) by the volume of our assays
(samples) in the PPO experiment ( 4 ml). Show work. Be sure to correct from
mmol.min-1 to umol.min-1 (1 pt).
(1) Enzyme activity = Vmax *volume of enzyme assay
4. Determine the specific activity of PPO in our assays (samples). This is calculated
by dividing the enzyme activity by the total number of mg of protein present in an
assay.
(2) Specific Activity = Enzyme activity/mg protein
Begin by calculating the amount of protein (mg) present in the portion of extract
used in our enzyme assays (0.2 ml). Use this value in equation (2) to determine
the specific activity of PPO. Show all work. Remember units (2 pt).
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