Biol3100

Colorimetric Assays, Standard Curves and Serial Dilutions

Often scientists want to determine the concentration of substances in solutions. They might need to
know the total amount of protein in urine, or glucose in blood. Biophotonics describes the
technology that focuses on the interaction of biological material with light and other forms of
radiant energy. Light can interact with biomolecules in several different ways: reflection,
absorption, transmission and light scattering. Colorometric assays take advantage of the light
absorbing properties of both biomolecules and dyes that interact with biomolecules. For instance,
protein concentration can be approximated by measuring absorbance at 280 nm. Which amino acids
are responsible for this absorption? However, not all proteins have similar light absorptive
properties. Instead, scientists routinely mix biomolecules with a light absorbing substance or dye for
concentration determination.

Colormetric assays are based on Beer’s Law: if a substance is dissolved in a transparent solvent,
the absorbance of the solution is proportional to the concentration of the absorbing substance.
A= Ecl
‘A’ is the absorbance, ‘E’ is the extinction coefficient defined as the absorbance of a 1 M solution
and is specific for each solution, ‘l’ is the distance of the light path (generally 1 cm in most
spectrophotometers) and ‘c’ is the concentration of the absorbing substance. The concentration can
be calculated if A, E, and l are known.

Thus, one can determine the concentration of a solution by comparing the absorbance to the
absorbance of known concentrations of the same solution. Beer’s Law, however, is only valid for a
range of concentrations and very high or low concentrations will no longer be proportional to
concentration.

There are three colorometric assays used for protein concentration determination. The Biruet
reagent is the least sensitive, but is often used in teaching labs. The Lowry reagent is the most
sensitive, but is also sensitive to interference from many common laboratory reagents and
chemicals, including detergents and many buffers. The Bradford assay is the most robust assay and
the one we will use today. The dye, Coomassie Blue G-250, binds to a number of the side chains of
amino acids in a protein: it interacts strongly with arginine through electrostatic interactions; the
aromatic rings of the dye stack with the aromatic rings of amino acids such as tryptophan; and the
dye weakly interacts with polar amino acids with hydrophobic R groups such as tyrosine. When the
protein binds the dye, the dye converts to a stable unprotonated, blue form. The intensity of the blue
is proportional to the concentration of the protein.

The absorbance of the unknown solution will be compared against a standard curve: a graph of the
absorbance of a series of known concentrations. The concentration of the unknown can be
extrapolated from the standard curve. In this lab, you will determine the concentration of protein in
some substances of unknown concentration.

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Materials
Polystyrene cuvettes
Protein Standards (BSA =bovine serum albumin)
Bradford reagent
Egg whites or milk or other liquid protein unknown
PBS (phosphate buffered saline) buffer

Procedure
1. Preparation of unknown samples
The unknown samples probably have too much protein and are out of the range of Beer’s
Law. These samples must be diluted, but by how much? Since the concentration is
unknown, a dilution series will be assayed so that at least one sample is within the linear
range of the standard curve. Generally, you might want to prepare a sample that is 2x too
high or 2x too low just in case. For this lab, dilute your samples 1:10, 1:100 and 1:1000. Be
sure to label your tubes appropriately!

2. Place 20 µl of each diluted sample into clean, labeled tubes.

3. Development of the Standard Curve

Using the chart below, you will prepare a series of samples of protein of known concentrations
referred to as “standard proteins”. Pipet 20 µl of each into a labeled tube. Calculate how much of
each protein you added to each tube.

Tube ul Standard mg of protein

mg/ml
1 20 µl PBS
2 20 µl 0.125
3 20 µl 0.250
4 20 µl 0.500
5 20 µl 0.750
6 20 µl 1.00
7 20 µl 1.500
8 20 µl 2.000
unknown 1:10
unknown 1:100
unknown 1:1000

4. Add 1 ml Bradford Reagent to all tubes, 1-8 and the 20 µl of each diluted unknowns. Mix.
The colorometric reaction should be complete after about 5 minutes, and the color should
remain stable for about 1 hour.

5. Instructions for Using the Spectrophotometer

After turning on the spectrophotometer allow the machine to warm up for at least 5 minutes before
making any measurements.
• Set the Wavelength: Set the wavelength using the nm ↑and ↓ buttons.

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 • Set the Blank: Before you begin making measurements of your samples fill the cuvette with
water or Bradford reagent (tube #1) and place in the holder being sure that the cuvette is
positioned so that the light path goes through the transparent part of the cuvette. Close the
lid. Press the 0% Abs/T button.
• Measure: Once the machine is blanked, you need only fill your cuvette with the solution to
be measured, close the lid, and read the absorbance.

6. Measure the absorbance of all samples at 595 nm. Pour each sample into the cuvette. If you start
with tube 1 and proceed, you need not rinse the cuvette between samples. Use the same cuvette for
all samples.

7. Make a standard curve by plotting the mg/ml of each protein standard on the x axis and the
absorbance of each protein standard on the y axis. Use a linear regression to get the equation for the
line. Use this line to determine the concentration(s) of your unknown(s).

Write-Up Instructions
Turn in the following as your lab report:
1. Data table that shows the absorbance of the standards and of the unknowns.
2. Properly labeled standard curve with a linear best fit line, provide the equation for the line.
3. Calculations
• Show the calculations, using the equation for the bestfit line, to determine the concentration
of protein in the diluted unknown.
• Show the calculation for determining the concentration of the undiluted unknown. Eg.
What is the concentration of protein in milk?
• Show the calculation of the concentration of unknown based on the carton nutrition label if
available.
4. Lab notebook pages
5. Answer the following questions:

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 1. What can you conclude about the protein profile of fat free vs whole milk?
2. What can you conclude about the protein profile of whole milk and soy milk?
3. What sizes are the predominant proteins in cow’s milk? Soy milk?
4. Why could your graph be plotted concentration (mg/ml) of protein vs absorbance OR mg of
protein vs absorbance? How would each plot affect your sample preparation and reading?

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Please attach the grading rubric to the front of your report.

Protein Assays Name:

Grading Rubric
_____ (4) Table of Absorbances (standards and unknowns)
_____ (4) Graph / Standard Curve
_____ (6) Calculations
_____ (4) Questions
_____ (2) Lab notebook

Total = _______ / ___20_____

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