DETERMINING THE CONCENTRATION OF THE UNKNOWN PROTEIN SOLUTION THROUGH BRADFORD ASSAY

AUTHORS
Marisse De Leon, Sarah De Los Santos, Heidy De Mesa, Estellito Dela Cruz, Gabrielle Delos Reyes Group 3, 2D-Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. The objective of the experiment is to determine the concentration of the unknown protein solution and to draw the standard curve by plotting the 595nm against a reagent blank. Standards were prepared by adding 0.3 and 0.4mL of BSA stock solution. Distilled water was added to each of the test tube to bring the volume to 1mL. For the determination of the unknown concentration, 1mL of the unknown protein sample was used. Through the use of the spectrophotometer, the absorbances (in nm) for the unknown proteins were determined. A standard curve was drawn by plotting the A595 versus the BSA concentration. The concentrations of the unknown proteins were solved by using linear regression. The result obtained for the concentration of the unknown for trials 1 and 2 are 106.117g/mL and 88.335g/mL. The average concentration is 97.226g/mL. The average absorbance is 0.2929nm

INTRODUCTION
There is no single protein assay method that yields absolutely accurate results. The only true andaccurate method for determining protein concentration is by acid hydrolyzing a portion of the sample and then carries out amino acid analysis on the hydrolyzate. But, this method is time-consuming. Eachmethod and assay has its own disadvantage and limitations.The most commonly used assays are the Ultraviolet Absorbance, Lowry Assay, BCA assay andthe Bradford Assay. The UV absorbance monitors the absorbance of aromatic amino acids, tyrosine andtryptophan or if the wavelength is lowered, the absorbance of the peptide bond. It is quick, with the samples that can be recovered. But, it is also highly susceptible to contamination by buffers, biologicalmaterials and salts. The Lowry Assay or enhanced copper since it reduces Cu +2 to Cu +1, sensitive over awide range and is the most commonly referenced procedure for protein determination but, it also takes aconsiderable amount of time. And the BCA assay or the bicinchoninic acid which is less susceptible toi n t e r f e r e n c e f r o m c o m m o n b u f f e r

substance and is very sensitive and rapid if you use elevated temperatures, but, the reaction does not go to completion when performed at room temperature and dilution is often necessary for concentrated protein samples The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed. During the formation of this complex, two types of bond interaction take place: the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure bind non-covalently to the non-polar region of the dye via van der Waals forces, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. The binding of the protein stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in solution is a measure for the protein