Download Protein Quantification:

Quantification of Protein by Using the BioRad Assay
Quantification of the amount of a substance in solution is a valuable skill in a biology lab.
Many procedures and experiments in biochemistry revolve upon knowing the
concentration of a substance in solution. For instance, a protein might be purified in a
biochemistry lab and its properties examined. In order to perform many of these
“downstream” procedures (experiments conducted with that substance after purification),
the scientist needs an accurate assessment of the concentration of the protein in solution.
The most common approach to determine the amount of protein in an unknown solution
is to compare the unknown solution to a series of known concentrations of a protein
similar to the one you are studying. This is the very essence behind generating a
Standard Curve. In essence, we are comparing an unknown to a set of references.
A simple illustration of this idea is detailed below:
Suppose the following boxes have a known “level of darkness” from 0-5:
0
1
2
3
4
5
These would be considered your “standards” or references because you know the value of
their darkness already.
Now you are given this box with an unknown level of darkness.
By eye, what would you say the level of darkness is? Perhaps the value is between 3-4 on
our scale. Essentially, what you have done is to create a standard curve in your mind and
you compared the levels of darkness using your eye to qualitatively, assess the unknown.
In biochemistry, we need to be more accurate than by eye. A useful tool that is used to
quantify substances in solution is the spectrophotometer. Every substance in solution,
due to its chemical properties, absorbs light best at a certain wavelength of light.
However, many substances are colorless in solution. So then, how do you quantify a
colorless substance in solution using a spectrophotometer?
The answer is an assay. An assay is a series of chemical treatments that are designed to
turn color in the presence of the substance you are examining. In our experiment today,
we will be using a protein, albumin, in concert with the BioRad Assay to develop the
color. The BioRad reagent in the assay is of a brownish color when no protein is present.
When one adds protein, the BioRad reagent turns blue. This color change can be detected
in a spectrophotometer at a wavelength of 595 nm. The more the protein the more
prominent the color change, the higher the absorbance in the spec.
Standard Procedures:
I.)
Preparation of the protein standards. ( Make them directly into the spec
test tubes)
The following table is for ease in preparation:
Concentration of
standard albumin
(mg/mL)
0 (Control)
0.5
1
2
3
II.)
Amount of 1
mg/mL stock
albumin
(ul)
0 ul
133 ul
267 ul
533 ul
800 ul
Amount of water
(ul)
Final volume of
standard (mL)
1600
1467
1333
1067
800
1.6
1.6
1.6
1.6
1.6
Assay procedure.
a.) Pipet, using a pipetman, 1600 ul, (800 ul 2x) of each standard to a cleaned
and labeled spec tube.
b.) Add an equal volume of the BioRad reagent to each tube. (Use a different
tip each time to eliminate cross contamination)
c.) Incubate at room temperature for about 5 minutes.
d.) Measure and record the absorbance at 595 nm in the spec. Be sure to blank
the spec with your water control.
III.)
IV.)
Generate a standard curve of your data (Abs @595 nm vs. Albumin
concentration in mg/mL)
Take 1600 ul of the unknown egg white and unknown yolk solutions and
repeat Part III. Calculate protein concentration of the unknowns from
the standard curve that you generated in part II.
What do I hand in?




Graph of Abs @ 595nm vs. albumin concentration
Calculation of unknown protein concentration.
One paragraph to address the following question: Am I sure that I am
correct in my determination of the unknown concentrations? Explain why or
why not.
What would you do if I gave you a very concentrated solution of albumin?
(One whose concentration is so high it pegs the spec) How could you
determine its concentration?