Revised Fall 2018

Biuret Protein Assay

Laboratory Safety
Lab coat, long pants, closed-toe shoes, safety goggles, and nitrile or latex gloves are required.

Learning Objectives
1. Identify methods to isolate protein from cells.
2. Understand how Biuret Reagent works to detect protein.
3. Understand the basics of spectrophotometry and use the spectrophotometer to determine the
absorbance of protein samples.
4. Describe the importance of a standard curve in scientific assays.
5. Assay known amounts of protein to create a standard curve and use the standard curve to determine the
amount of protein in an unknown sample.

Introduction
Proteins are one of the major classes of biological molecules. They are found in all cellular compartments
and are instrumental in almost all of the functions of the cell. In order to study these important
macromolecules, scientists often need to determine the amount of protein in specific experimental samples.
There are a number of very good and very sensitive protein assay methods, each with its own advantages
and disadvantages. One of the simplest and most common is the Biuret Protein Assay. It is inexpensive, easy
to prepare and use, but is not as sensitive as some of the other methods.

The basis of the Biuret Protein Assay is the ability of amide groups in proteins to
complex with copper ions at a basic pH. Remember that the primary structure of
proteins consists of amino acids joined together by peptide bonds. When those
peptide bonds form, the amino group (-NH2) becomes an amide group (-NH). In the
biuret reaction, four amide groups complex with copper (see figure).
The biuret reagent is blue, but when the copper complexes with the amide groups,
the color changes to violet. The intensity of the color produced is directly
proportional to the number of peptide bonds participating in the reaction and thus
in the amount of protein present. Note that this assay will only detect polypeptides that are 3 amino acids
in length or longer.

The intensity of the color produced by this assay can be measured using a spectrophotometer. The
spectrophotometer is an instrument used to
measure the amount of light absorbed by a
solution. When light passes through a sample,
some of that light is absorbed, whereas some
is transmitted. Each molecule has a
characteristic absorption spectrum,
wavelengths of light that it absorbs. For example, the wavelength of light best absorbed by DNA is in the
260nm range. The complex formed in the Biuret Assay absorbs light best at 550nm. The spectrophotometer
works by taking white light and splitting it into separate wavelengths. Specific wavelengths can then be
directed towards the sample. On the other side of the sample is a detector (a photocell) that determines
the amount of light that was transmitted through the sample. From that information, the spectrophotometer
calculates the amount of light that was absorbed. The reading you get is the absorbance. There is a direct
relationship between the concentration of your sample and the absorbance, so the higher the concentration,
the higher the absorbance. For colorimetric assays, like the Biuret Protein Assay, there is also a direct
relationship between the intensity of the color and the concentration.
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Before the amount of protein can be determined

using the method, the protein must first be isolated
from the cell. Remember, proteins are ubiquitous,
that is they are found in all cellular compartments,

https://www.ncbi.nlm.nih.gov/books/NBK26878/figure/A1903/
including both the cytosol and the membrane. To gain
access to the hydrophilic proteins inside the cell as
well as solubilize the transmembrane proteins,
scientists typically use a detergent as well as
mechanical disruption. Detergents, such as SDS
(sodium dodecyl sulfate), are amphipathic, much like
phospholipids, although they have only a single tail.
The detergents tend to form micelles when placed in
an aqueous solution. When a detergent comes in
contact with a membrane, the hydrophobic tails bind
the hydrophobic regions of integral membrane
proteins, and displaces the adjacent lipid molecules.
The part of the detergent that is polar, brings the membrane proteins into solution. In addition, the displaced
phospholipids form micelles with the detergent molecules, such that the lipid bilayer does not reform.

Standard curves are essential components of many biomolecular techniques used to determine the
concentration of an unknown substance. In the biuret assay, a standard curve is generated to calculate the
concentration of protein in a sample. A standard curve consists of samples (the standards) with a known
concentration which are measured for their optical absorbance values using a device such as the
spectrophotometer. The known protein concentration and optical absorbance values for these standards
can be plotted on a graph, creating a standard curve. The standard curve displays the relationship between
protein concentration and optical absorbance and is then used to determine the protein concentration of an
unknown sample. The optical absorbance value of an unknown sample may now be compared to the plotted
standard curve and a concentration of protein for that sample can be determined.

Activity 1 – Determination of protein amount from a food sources

We know that proteins are critical to the functioning of the cell. We also know that proteins are built from
amino acids. Amino acids can be synthesized de novo (from scratch) in the body or obtained from the diet.
Essential amino acids cannot be synthesized in the body and can only be obtained from outside sources.
Thus, proteins are an integral component of the diet. Table 1 shows the protein content information that
you might find on a nutrition label for various foods.

In this experiment, we will isolate protein from a food source and determine the protein per gram of sample.
The experiment will be broken into 4 parts: isolating protein from your samples, preparing a standard curve,
adding the biuret and reading the absorbances, and graphing the standard curve for use in determining the
concentration of protein in your samples. The graph may be created in lab or as a post-lab activity, depending
time as well as your instructor.

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Activity 1A – Isolation of protein from “unknown” samples

Experimental Protocol
1. Each group will choose one of the food samples from those available to test. Record your
choice in Table 2.
2. Once you have chosen your sample from which you will be isolating protein, form a hypothesis
as to how much protein you think you measure per gram of food based on the information in
Table 1 above. Record your hypothesis here: _______________________________________
___________________________________________________________________________
3. For a solid food sample weigh out approximately 0.2g of sample into a labeled 50ml blue cap
conical tube. Record the exact weight in Table 2. For a liquid food sample pipette 200µl of your
sample into a labeled 50ml blue cap conical tube. Record the exact weight in Table 1.
4. To your sample, add 10-15 colored glass beads and 10ml of 0.5% SDS.
5. For liquid food samples, vortex the mixture for 10 seconds then let the samples sit at room
temperature for 10 minutes.
6. For solid food samples, vortex the mixture for 1 minute, then centrifuge the mixtures at
2500rpm for 8 minutes. Centrifugation will cause all of the cellular components and other debris
to settle at the bottom of the tubes. The protein will be in the supernatant (the liquid at the
top).
7. While the samples are centrifuging or incubating on your benchtop, move on to activity 1B.

Table 2. Unknown Sample

Food type? Amount weighed out (g)

Activity 1B – Preparation of the standard curve

The standard curve will be made using known amounts of the protein BSA (bovine serum albumin). Each
concentration will be run in duplicate to ensure proper pipetting.

Experimental Protocol
1. Label two sets of test tubes with the numbers 1-6. The
numbers should be written at the top of the tubes, not at the
bottom.
2. Obtain a tube of the BSA stock solution from the front. The
stock solution is 10mg/ml.
3. Pipet the amount of water indicated in column 3 of Table 3
into each tube. For example, you will pipet 1ml into Tube #1.
4. Pipet the appropriate amount of BSA stock solution into
each tube (column 4 of Table 3).
5. When you have finished, all of your standard tubes should
contain a volume of 1ml.

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Activity 1C– Performing the Biuret Protein Assay

Experimental Protocol
1. Label the tops of two tubes for your food sample.
2. Carefully remove your sample from the centrifuge, being careful not to shake the sample and
disrupt the supernatant.
3. Pipet 1 ml of the supernatant from your sample into each of the food sample tubes
4. Add 2ml of biuret reagent to all of your standard tubes and sample tubes, 14 tubes total.
5. Cover the tubes with parafilm and briefly vortex to ensure that the sample and biuret are
thoroughly mixed.
6. Incubate the tubes at room temperature for 15 minutes.
7. While the tubes are incubating, turn on the spectrophotometer. Once it has warmed up and is
ready, adjust the wavelength to 550nm. Check with your instructor if you are unsure how to do
this.
8. After the incubation, you will read the absorbance of your samples. To do this open the lid of
the spectrophotometer. Hold the first test tube (tube #1 0mb/ml protein) by the top. Gently wipe
the bottom of the tube with a Kimwipe. Place the tube into the spectrophotometer and gently
close the lid. DO NOT slam the lid shut.
9. The first test tube (tube #1) will be used to blank the spectrophotometer. This is much like taring
a balance. To do so, you will push the zero/blank button once tube #1 is in the spectrophotometer.
This sets the absorbance to zero.
10. Remove test tube #1 and measure the absorbance for your duplicate standard tube with
0mg/ml protein. Do not zero the instrument. The absorbance should read close to 0 if not please
call over your instructor. Measure the absorbance of all of your other tubes.. Record the
absorbances in Table 4.

Table 4. Absorbances of Standards and Unknowns

Conc. Conc.
ABS ABS ABS average
(mg/ml) (mg/g)
Standard 1 0

Standard 2 1

Standard 3 2

Standard 4 3

Standard 5 4

Standard 6 5
Estimated =
Unknown A

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Activity 1D – Graphing the standard curve and determining protein concentration of unknowns
1. Using the BSA concentrations (column 5) and average absorbances (column 4) from Table 4, plot
the standard curve on the graph paper included here.
2. Use the graph and your unknown absorbances to estimate the protein concentrations in your
unknown samples.
3. Record the values in Table 3, in the concentration column.
4. Convert your mg/ml measurements to mg/g and record the data in Table 3. To do this use the
example below:
 Multiply the mg/ml measurement by 10 ml to get the total mg measured. Divide that
number by the exact number of grams you weighed out at the beginning of the
experiment.
 Example: you originally weighed out 0.4 g of tofu and your calculated protein
concentration is 3.2 mg/ml. To get the mg/g measurement:
3.2 mg/ml * 10ml = 32 mg
32 mg/0.4 g = 80 mg/g

Based on your calculations, what is the protein content (per gram) of your food sample?
_______________________________________________________________________________
How does this compare to the protein content that you might find on a nutrition label (Table 1)?
_______________________________________________________________________________
_______________________________________________________________________________
Was your hypothesis supported?
_______________________________________________________________________________
_______________________________________________________________________________
Why might the protein content you measured differ from the reported values on Nutrition labels?
(think about the purpose of each step of the protocol and how this impacts the final protein value
calculated) ______________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________

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Pre-lab Activity
Prior to this lab you should answer the following questions:
1. What monomers are proteins built from? _______________________________
2. What component of biuret reagent forms a complex with amide groups and changes color? __________
3. A sample with a high color intensity would be expected to have a _______________ amount of protein
present.
4. What is the name of the machine that you will use to measure the amount of light absorbed by your
samples? __________________________
5. The weight of different numbers of m&m’s was recorded and the data presented in the graph below. If
you were given a bag of m&m’s how could you determine the number of m&m’s in the bag without
opening it? ______________________________________________________________________________
How many m&m’s would you predict you would have in a bag that weighed 8g? _____________________

number of
weight (g)
m&m's
0 0
1 0.9
5 4.5
10 9.1
15 13.2
20 18
25 22.4

6. The above graph is an example of a standard curve and the method that we will use in lab to determine
protein content in a sample of food.
What will be the axis labels on the standard curve you will create in lab? (i.e. what are you measuring?)
_______________________________________________________________________________________
_______________________________________________________________________________________
Do you think that there is a standard curve graph for the biuret protein assay that you can look up and use
instead of running one in lab? ______________________________________________________________
_______________________________________________________________________________________

Post-lab Activity
You may be required to submit an assignment based on this lab. You instructor will provide you with the
details required for your submitted assignment.