Protein Quantitation using the Bradford assay and Spectrophotometry
Protein quantitation is a study which is used to determine protein concentration in an unknown sample by comparing its assay response to one which is already known. This has uses in a broad range of research topics which include protein analysis. Two bovine serum albumin (BSA) dilutions of unknown concentrations using the Bradford assay method of protein quantification were used. BSA is a fatty acid carrier protein in the blood; this is used as it stabilizes enzymes and has advantageous properties such as protection from oxidative damage and stabilization of proteins for analysis.
The Bradford assay method is one which is regarded as a relatively quick and simple process, and the results helped to determine its accuracy (Bradford, M. 1976). Important aqueous solutions used throughout the experiment were distilled water, Coomassie brilliant blue G-250 (CBBG) and BSA. The acidic CBBG dye was used to stabilize the binding of this to the BSA, therefore increasing the maximum absorption from 465nm to 595nm (Spector, T. 1978).
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The spectrophotometer is an absorbance measuring instrument that produces light through two bulbs; the first a visible light and the second one which produces an ultra violet light. Five known substances from 0. 2 mg/ml to 1. 0 mg/ml, one blank sample and two unknown were used to be tested. In the beginning these solutions were already in 1. 5ml microfuge tubes, the amount of stock needed for each was then calculated with the equation C1V1 = C2V2.
C1 being the concentration, V1 the value of the starting solutions, C2 the desired concentration of the final stock and V2 the volume of the final stock. Using pipettes, the proportionate amounts mixed with distilled water were used and placed in separate falcon tubes. The concentrations were then mixed with a 5. 0 ml of diluted dye reagent, followed by a five minute incubation period. The solutions were each pipetted into cuvettes and placed in the spectrophotometer which was set at 595mm.
The blank sample was the first to be examined as this produces an indication of background absorption. The readings were given and entered into a curved scatter graph comparing absorbance against concentration. By adding a trend line to the plotted graph with the equation y = 1. 0471x + 0. 528 and R2 = 0. 8421 the results were found, with the values from the spectrophotometer. The equations to find the results were unknown 1 (0. 840 – 0. 528) / 1. 047 = 0. 298 mg/ml and unknown 2 (0. 850 – 0. 528) / 1. 047 = 0. 307 mg/ml.
These results can be said to be successful as it shows that the first unknown concentration was accurate with a 0. 002 mg/ml difference. The second unknown concentration had a difference of over double its value. The results show that since they both used the same method and the first unknown was precise that the proceedings may have been carried out correctly and the significant reason for variation for the second unknown was that it may have been affected by human error. This was primarily through bad pipetting technique during dilutions, and incorrect handling of the cuvette.