Protein Assays

Protein assays are used to estimate the protein concentration in a given sample.
Below is some information about both of the assays you will be using in the lab on
Thursday.

1. Biuret assay

The biuret method depends on the presence of peptides bonds in proteins. When a
solution of proteins is treated with cupric ions (Cu2+) in a moderately alkaline
medium, a purple coloured Cu2+ - peptide complex is formed which can be
measured quantitatively by spectrophotometer in the visible region. Biuret reagent is
light blue. Peptides and proteins (long-chain polypeptides) react with Cu2+ in an
alkaline environment to create a blue-violet colour.

Diagram of the biuret reaction. By reducing the copper ion from cupric to cuprous form, the reaction
produces a faint blue-violet colour

The intensity of the colour produced is proportional to the number of peptide

bonds that are reacting, and therefore to the number of protein molecules present
in the reaction system.

In this experiment you will be making and using the standard curve of bovine serum
albumin (BSA). A standard curve is a quantitative research tool; it is a method of
plotting assay data that is used to determine the concentration of a substance like
protein. First you perform a biuret assay with various known concentrations of bovine
serum albumin. The response is absorbance of the coloured product. You use these
data to make your standard curve, concentration on the X axis, and the absorbance
on the Y axis. You are also performing the biuret assay with two samples (tubes 7&8
- gelatin, and tubes 9&10 – globulin). You want to know the concentration of the
protein in each sample. To analyze the data, fit a line or curve through the standard
curve. For the samples read across the graph from the absorbance on the Y-axis
until you intersect the standard curve. Read down the graph until you intersect the X-
axis. The concentration of your sample is the value on the X-axis.

2. Lowry Assay

The phenolic group of tyrosine and trytophan residues (amino acid) in a protein will
produce a blue purple colour complex, with maximum absorption in the region of
~660 nm wavelength, with Folin- Ciocalteau reagent which consists of sodium
tungstate molybdate and phosphate. Thus, for this assay the intensity of colour
produced depends on the amount of these aromatic amino acids present in your
protein and will thus vary for different proteins. The intensity of the colour
produced is proportional to the number of tyrosine and tryptophan amino acid
residues that are reacting. Again your standard curve is calculated use Bovin
Serum Albumin (BSA). The chemistry for this equation is more complex than the
Biuret assay and is shown schematically below.

For this assay you will make a standard curve, concentration on the X axis, and the
absorbance on the Y axis. You will also be performing the Lowry assay with two
samples (tubes 7&8 - gelatin, and tubes 9&10 – globulin). You want to know the
concentration of the protein in each sample. To analyze the data, fit a line or curve
through the standards. For the samples read across the graph from the spot on the
Y-axis that corresponds to absorbance of the sample until you intersect the standard
curve. Read down the graph until you intersect the X-axis. The concentration of your
sample is the value on the X-axis