Bio 109 Lab 4 report.pdf - Lab 4: Quantitation of Protein...

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Lab 4: Quantitation of Protein
GOALS:
• To acquaint students with the Bradford test for protein quantitation
• To introduce students to the basics of spectrophotometry
• To introduce students to the use of a spectrophotometer
• To teach students how to prepare dilutions for analysis
• To teach students how to construct a standard curve
• To teach students how to utilize spreadsheet software to organize,
display, and analyze data
INTRODUCTION
It is impossible to place biological material under a microscope and count
the number (quantity) of protein molecules per unit volume the way we
can count the number of cells. Therefore, something measurable that is
proportional to the concentration of the substance of interest must be
identified.
Beer's law states that when a solute absorbs light of a particular
wavelength, the absorbance is directly proportional to the concentration
of the solute in solution. The measurement most commonly used in protein
assays is the absorbance of light. However, proteins do not absorb
sufficient light to assay - by themselves.
The Bradford assay uses a dye, Coomassie Brilliant Blue G-250, which was
first described by Dr. M. Bradford in 1976. This assay takes advantage of
the chemical properties of the dye and the dye’s ability to interact with
the side chains, or R-groups, of specific amino acids. Coomassie G-250
exists in multiple forms. As part of the Bradford solution, the dye exists in its
cationic state and takes on a reddish-brown color. The peak absorption
of the dye in this state is 470 nm. When the dye binds to and interacts with
amino acids in proteins, the dye is converted to a stable unprotonated
blue form, and the absorption maximum shifts from 470 nm to 595 nm. The
dye binds to proteins using three types of interactions. The primary
interaction of the dye with proteins occurs through arginine, a very basic
amino acid, which interacts with the negatively charged sulfate groups
through electrostatic interactions. Other weaker dye-protein interactions
include the interaction of the aromatic rings of Coomassie G-250 dye with
the aromatic rings of amino acids, such as tryptophan, through electron
stacking interactions. Finally, the dye also weakly interacts with polar

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amino acids that have hydrophobic R-groups, such as the aromatic ring
of tyrosine.
The exact chemical interactions or binding properties of Coomassie G-250
dye are illustrated below.

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This stable blue form of the dye is easily observed and quantified in a
spectrophotometer. In accordance with Beer’s Law, there is a correlation
to the amount (absorbance) of blue color and the amount of protein
(solute) in the sample. The more protein, the more intense the blue color.
The simplicity of the assay allows the results to be measured qualitatively
by eye, or quantitatively with a spectrophotometer.