CHAPTER TWO
2.2 Protein quantitation assays
The simplest and most widely used method for protein determination is a spectrophotometer method measuring the absorbance of a protein at 280 run (A28o) and calculating protein concentration using the extinction coefficient of that protein. Most proteins show maximal absorption at 280 run due to the presence of aromatic amino acids, such as tyrosine and tryptophan. Different proteins have different ratios of aromatic amino acids, therefore, in order to use absorbance at 280 run, proteins must be pure and of known extinction (absorption) coefficients. For IgG and IgY antibodies their extinction coefficients are 1.43 and 1.25 ml/mg/cm, respectively (Dennison, 2003).
For pure protein preparations with known extinction coefficient, the concentration using a cuvette with a 1 cm path length was calculated according the following formula.
A=ExC
Where A= Absorbance at 280 run.
E = Extinction coefficient (ml/mg/cm) i.e.(1 mg/ml of 0.1% solution) of the test protein in a 1 cm cuvette.
C = protein concentration in mg/ml
2.2.1 Bradford dye binding assay
As it is impossible to determine the concentration of a protein mixture using an extinction coefficient, Bradford (1976), therefore, developed a method which determines the total protein concentration using a dye-binding assay in which the binding of a dye to a protein causes a shift in the absorption maximum. For Coomassie brilliant blue G-250 the shift is from 465 run to 595 run allowing monitoring of absorption shift at 595 run.
This method for quantitation of proteins is still the best for protein measurements. Many authors have come up with slight modifications but the Bradford assay remains widely used because of its ease of performance, rapidity, relative sensitivity, and specificity for proteins (Zor and Selinger, 1996).
Bradford's Coomassie brilliant blue G-250 protein-binding dye exists in three forms: red (cationic), green (neutral), and blue (anionic) which absorbs at 470 run, 650 run and 590 run, respectively. As the binding of dye to protein ratio increases the blue colour increases and the red colour decreases.Due to the overlapping absorption characteristics of the red and blue dye forms the method gives a slight non-linearity (Bradford, 1976).
To avoid this non-linearity Zor and Selinger (1996), recommended reading the absorbance at 590/450 run so that protein concentration may be established by measurement of the increasing dye absorption at 590 run and decreasing dye absorption at 450 run. This ratio was found to be directly proportional to the concentration of protein present and gave a more linear curve than Bradford's method, increasing
sensitivity 10-fold and allowing quantitation of down to 50 ng of a standard protein.
Measuring at 590/450 nm also decreased the interference due to competition of low amount of SDS with the dye.
Another modification was introduced by Read and Northcote (1981). These researchers observed that increasing the amount of dyein assay solution or decreasing the amount of phosphoric acid and replacing Coomassie brilliant blue R-250 with Serva Blue G-250 increases sensitivity and also adjusts the colour range achieved with proteins to values close to that seen for equivalent concentration of the standard protein BSA. BSA is a commonly used standard protein in the Bradford assay as it has high colour yield unlike other standards which almost have the same absorbance as the proteins for analysis (Zor and Selinger, 1996). The modification of Zor and Selinger (1996) and Coomassie brilliant blue was replaced with Serva Blue in the study as the increased amount of dye used in the Read and Northcote (1981) method has previously shown to have a precipitating effect.
2.2.1.1 Reagents
Bradford dye reagent. Dye reagent was prepared according to Bradford (1976) with the exception that Serva Blue G-250 (50 mg) was dissolved in 25 ml 95% ethanol and used instead of Coomassie blue. A volume of 50 ml phosphoric acid [85% (w/v)] was added and the solution was diluted to 500 ml with double de-ionized water (ddH20 ). The solution was stirred for 30 min and filtered twice. The final concentration of the reagent was 0.01% (w/v) Serva Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid.
Standard BSA solution Cl mg/m}). BSA (Fraction V) (10 mg) was dissolved in 10 ml ddH20 to make a 1 mg/ml solution.
2.2.1.2 Procedure
Stock bovine serum albumin (BSA), prepared as in the above, was used to prepare standard curves for the microgram and nanogram range of protein. Five replicates of 40 Ills of the diluted sample were dissolved in 160 Ills of ddH20 each. Each sample (200 Ills) was mixed with 800 Ills of Bradford reagent in 1.5 ml Eppendorf tubes to make up 1 ml. The tubes were gently vortexed and transferred to clean plastic cuvettes. [Only plastic cuvettes were used in the experiment as Coomassie brilliant blue dye binds to glass cuvettes and gives erroneous readings (Bradford, 1976)]. Dye-protein mixtures were allowed to react for two min and absorbance of each sample was measured at 590 run and 450 run within one hour. Double de-ionized water (ddH20) served as a blank instead of dye reagent as in the conventional Bradford's assay because the free dye is also measured at 450 run thus, no free dye control is needed. The ratio ofAS90/4S0 was calculated and a standard curve was plotted against the amount of protein applied. A graph was constructed in Microsoft Office Excel software and a linear regression equation was calculated to determine the concentration of protein.