The McGill Physiology Virtual Lab

Biochemical/molecular techniques

Background concepts:
Spectrophotometry and protein concentration estimation

A spectrophotometer works by shining a light beam through a solution containing an analyte (or molecule being studied). Light absorption is detected using a photosensitive element which reads out optical density (O.D.).  Estimation of the concentration of the analyte by spectrophotometry is possible because the quantity of light absorbed by the analyte in a solution (or O.D.), increases linearly with concentration.
The technique
The spectrophotometer is a device which measures the concentration of a substance (analyte) in solution. The analysis carried out by a spectrophotometer is based on Beer's Law which relates the amount absorbed by a substance to its concentration in a solution. The concentration of an analyte is directly proportional to the amount of light absorbed by the solution, and inversely proportional to the logarithm of the amount of light transmitted by the solution.

Beer's law is followed only if the light entering the solution is composed of a single wavelength. This light set at a specific wavelength enters the cuvette which contains the solution to be tested. Some of the incident light (I0) is absorbed by the solution - the amount absorbed depends on the concentration of the solution - and the rest of the light (the transmitted light I) is detected. The ratio between the amount of light that goes into the solution and the amount of light that leaves the solution gives the absorbance (A) of the solution: A= -log (I/I0). This definition supposes that all the incident light is either transmitted or absorbed, reflection or scattering being negligible.
The following equation: A=
elc ,
where
e is the substance and wavelength specific absorption coefficient,
I is the length the light travels through the sample
and c is the concentration of the sample,

shows the relationship between the absorbance and the concentration of a substance.

The standard curve

Since the samples are tested under the same condition: under a set wavelength (which is the analytical wavelength: the wavelength of maximal absorbance chosen from the absorbance spectrum of the substance) and incident light distance, e and l are the same for all samples, thus A varies linearly with c.
The first step is to construct a curve relating known analyte quantities to their measured absorbance. OD recorded from the spectrophotometer (absorbance) is plotted on the Y-axis and the analyte amounts are plotted on the X-axis. A series of solutions with known analyte quantity (the "standards") are prepared, the absorbance values are recorded and plotted versus the solution amounts; a straight line can be best fitted. The first spectrophotometer cuvette is the "blank": it contains no analyte, the OD is recorded.

Example of a calibration curve constructed with a set of Bovine Serum Albumin (BSA) standards; note that the first standard contains all reagents except the protein (BSA):

 Absorbance (OD) Quantity BSA (mg) 0.598 0 0.648 2 0.745 5 0.927 10 1.123 15 1.225 20

Next, the absorbance of the unknown samples is measured. The absorbance value will correspond to a value on the calibration graph or standard curve.

The Bradford method of protein quantification

Assays which generate a protein-dependent colour change, are measured by spectrophotometry: Beer's law can be applied here for accurate quantitation of protein by selecting an appropriate ratio of dye volume to sample concentration. Over a broad range of protein concentrations, the dye binding method gives an accurate but not entirely linear response.

The Bradford assay is mediated by the Coomassie Blue G-250 dye.
As with any colorimetric protein assay, there is variation in colour response to different proteins. Both the colour change due to dye binding and the variation in colour response can be attributed to the dye having three absorbing species:  a red cationic species, a green neutral species, and a blue anionic species. At the assay pH (under strongly acidic conditions) the dye molecules are doubly protonated and are present as the red cationic dye form. Binding of the dye to protein stabilizes the blue anionic dye form, detected at 595 nm.

Coomassie brilliant Blue G-250: the higher degree of conjugation (single/double bonds), the higher the absorption of light. The Coomassie blue reagent has been shown to interact mainly with arginine residues, but weakly with histidine, lysine, tyrosine, tryptophan and phenylalanine residues; VanderWaals forces and hydrophobic interactions also participate in the binding mechanism. The number of Coomassie reagent ligands bound to each protein molecule is approximately proportional to the number of positive charges on the protein (1.5 - 3 dye molecules/charge).

The assay is also influenced by non-protein sources (e.g.: detergents) and becomes progressively more non-linear at the high end of its useful protein concentration range: interference from non-protein compounds is due to their ability to shift the equilibria among the three species of the dye. The assay is also protein-dependent (different proteins have different amino acid compositions) and varies with the composition of the protein.
These limitations result in the necessity of having the appropriate protein standard solution; to get the most accurate results, the standard must be composed of a mixture of proteins as similar as possible to the unknown. Bovine gamma globulin or bovine serum albumin may be used as standards. The standard will give a colour yield similar to the protein being assayed. The assay can be used with samples having protein concentrations between 2-20
mg/ml.