Laboratory 7

Biuret Protein Assay

Found on page 91 of Lab Manual

 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.
 Proteins

• Major class of biological molecules

• Many, many different kinds of proteins, each with
specific structure and function.
• Important in almost all aspects of cell functioning

Hemoglobin GLUT5

https://pdb101.rcsb.org/motm/41 https://pdb101.rcsb.org/motm/208
 Proteins

• Made of amino acids joined by peptide bonds

• Carboxyl group becomes carbonyl group

• Amino group becomes amide group
 Black = carbon
Red = oxygen

Proteins White = hydrogen

Blue = nitrogen
Green = Side chain/
R group
 Biuret Protein Assay
• To study proteins, often need to quantitate amount
of protein in a sample
• Numerous protein assay methods and reagents
• Each method has its own advantages and
disadvantages, and no one method can be
considered the best
• Biuret Protein assay is inexpensive and easy to
prepare and use
• Biuret Protein assay is less sensitive than other
methods of protein detection
 Biuret Protein Assay
• The basis of the Biuret Protein Assay is the ability of
amide groups in proteins to complex with copper
ions at a basic pH.

• Contains sodium hydroxide (NaOH) and copper (II)

sulfate.
• Four amide groups are required to complex with
copper, so must have at least 3 amino acids per
polypeptide
 Biuret Protein Assay
• Colorimetric assay
• Color changes from blue to violet when proteins
present
• Intensity of color produced is directly proportional
to the amount of protein present

A B C D E
 Spectrophotometry -
Quantifying the Color Change
• Need to measure intensity of color to determine A B
actual protein concentration

• The complex formed with the Biuret assay has a

unique wavelength of light that it absorbs

• The spectrophotometer is an instrument used to

measure the amount of light absorbed by a
solution.
 Spectrophotometry -
Quantifying the Color Change
 Protein Isolation from Cells
• Proteins must be isolated from cells

• This is achieved using mechanical disruption and

detergents such as SDS (sodium dodecyl sulfate)

https://www.ncbi.nlm.nih.gov/books/NBK26878/figure/A1903/
 Standard Curves –
Quantifying the Amount of Protein
A B

• A standard curve must be used to determine the

exact concentration of protein in a sample.

• Standard curves use the measurement of a known

quantity to determine the amount of an unknown
https://www.youtube.com/watch?v=0luczWOo0rQ
 Determination of Protein
Amount from Food Sample

• Fill in the table

• Form a hypothesis
 Determination of Protein
Amount from Food Sample

1. Isolation of protein from “unknown” samples

2. Preparation of standard curve

3. Performing the Biuret Protein Assay

4. Graphing the standard curve and determining

the concentration of the “unknown” samples
Isolation of protein from
“unknown” samples
Preparation of standard curve
Performing the Biuret Assay

Add 2ml to each tube

Performing the Biuret Assay
Graphing the Standard Curve and
Determining the Protein X-axis
y-axis
Concentration of Unknowns
Post-Lab Activity

• Individual lab reports

• Details will be provided by your lab instructor

Don’t forget your PPE!