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The Bradford Protein Assay is a spectroscopic procedure of analysis that helps in the determination of the concentration of proteins in a sample solution. A quicker, simpler and safer procedure used in finding the protein concentration in an aqueous solution was developed in the 1970s by Marion Bradford, a biochemist. The procedure has been found to be very efficient thus has become a preference displacing the other methods including Biuret and Folin, which were originally used in the measurement of soluble proteins (Freshney, 2011, p.159). Because of the disturbance created by the detergents that have been used in the solubilisation process of the membrane-bound proteins, this procedure is not suitable in the form described in this experiment.
The original publication of the Bradford Reagent protocol was done in an article Analytical Biochemistry. The protocol underwent refined modification that made it more useful especially for membrane-bound proteins. The modifications were published in the same journal article in the preceding years. The Bradford protein assay is a very convenient method of determining the concentration of proteins (David W. Burden, 2012, p.212). It supplies the dye reagent used in the estimation of the protein concentration at 1x concentrations well as two protein assay standards at prediluted concentrations of seven.
The seven prediluted standards are efficiently packaged in screwcap vials of volume 2ml. The packaging reduces and eliminates any form of wasteful sharp ampoules. The packaging also ensures the protein is stable over the product's shelf life. Bradford Protein Assay measures the concentration of proteins in a food sample by adding Coomassie dye to the food sample under acidic conditions. A colour change from brown to blue is noted when proteins in the food sample bind with the Coomassie dye. The concentration or level of the blue colour can be determined using spectrophotometer hence determining the protein concentration in the food sample (Copeland, 2013, p.251).
Among the advantage of Bradford Protein Assay are the few steps involved, no need of heating as well as its ability to provide a calorimetric response of greater stability. The response of Bradford Protein Assay method is affected by non-protein sources. It tends to be more nonlinear towards the greatest concentration of protein range (Freshney, 2011, p.266). The response of this method is also affected by proteins changes with a change in the composition of the protein being tested. Due to these limitations, there is need to standardize the solutions of proteins to achieve results that are more accurate.
The amount of light absorbed by a sample is measured by the use of a spectrophotometer. This instrument concentrates a beam through a sample thereby taking measurements of the light intensity that reaches the detector (Blankenship, 2012, p.118). The beam of light is composed of a stream of photons, which are absorbed by an analyte molecule when they come across each other. The intensity of the light beam is reduced as more photons are absorbed by the analyte material during encounters.
The objective of this experiment is to find the concentration of the unknown solution of protein and draw the standard curve by plotting the 620 nm against a reagent blank.
The procedure used in the determination of the presence of protein using Bradford Protein Assay for the case of this particular experiment was slightly altered with some of the steps skipped, and others changed to suit the nature of the experiment. The method used was as shown below
Table 1 shows the absorption levels for the two attempts of the experiment and the mean absorption of DCIP at different percentage concentrations when diluted.
The graphical illustration of the tabular results is as shown in the line graph below. A line of best fit was obtained when the results were plotted on a graph.
Bradford Protein Assay is a colorimetric protein assay, which is dependent on the absorbance shift in Coomassie dye. It is expected in this test that the Coomassie dye shifts the colour from brown to blue to illustrative the presence of proteins in a sample solution. The blue colour change results from the binding of the dye present in the sample solution (Bisswanger, 2013, p.122). Two types of interactions of bonds are observed during the formation of this complex compound. The first interaction is the donation of a free proton by the brown/red Coomassie dye. The dye donates its proton to the ionizable group that is on the protein and such a donation results into a disruption of the native state of the protein thereby leading to the exposure of its hydrophobic pockets. The exposed pockets, which are on the tertiary structure of the protein non-covalently, bind to the region of the dye that is not polar by Van der Waals forces. In so doing, the favourable amino group of the protein is brought close to the negative charge of the dye (Ganapathy-Kanniappan, 2018, p.351). The ionic interaction between the amino group and the dye is made stronger by ionic interaction between them. Through binding of the protein, the blue form of Coomassie dye achieves stability hence the quantity of complex available in the solution is determined by the concentration of the protein using the reading of the absorbance.
Fig.1. Structure of Coomassie Brilliant Blue
The extinction coefficient remains constant for the complex solution of the dye and protein for over 10 times the range of concentration (Becker, 2012, p.148). This makes Bradford protein assay very useful. The concentration-absorbance curve can be changed depending on hydrophobic or arginine amino acids percentage in each of the proteins. This is because Bradford protein assay takes measures of the number of residues of arginine or hydrophobic amino acids. This leads to the need of a standard having a protein having protein proximity to the measured protein in the composition. An example of such standards is Bovine Serum Albumin (BSA). The dye reagent is found to react more with residues of arginine, and the rate of reaction is found to be lowered with residues of tryptophan, lysine, phenylalanine, tyrosine and histidine. The accuracy of the assay is considerably lower when measuring basic or acidic proteins (Hock, 2015, p.215). The sensitivity of Bradford to BSA is relatively higher than with average proteins by approximately a factor of two. 1 M NaOH is added to facilitate solubility of the protein membranes and also reduce the variations in the yield of colour between a protein and another protein. Immunoglobin is the most preferred standard of protein.
Any error in the experiment can be a cause of alarm and may interfere with the whole experiment and its results. It is for this reason that care is taken such that the prediluted standards are packaged conveniently to eliminate any wasteful and sharp ampoules as well as keep the shelf life stable (Bisen, 2014, p.188). Care should be taken when pipetting to avoid such errors as having inadequate or excess of the reagents or the dye. It is also important to have the spectrophotometer zeroed using the regent blank as it may as well be a source of error in the experiment. Another precaution is performing not less than two reagents blank or instead use a water buffer.
The absorbance values that were duplicated in the experiment were close, and the graph turned out to have a line of best fit. The only significantly different in the values of absorbance was observed at 0.002% of DCPIP. From the obtained results, it can be understood that the values have a major increase from Dilution B to Dilution A. this is evidently illustrated on the graph from the huge shift between 0.001% DCPIP concentration to 0.002% DCPIP concentration.
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Bisen, P.S., 2014. Laboratory Protocols in Applied Life Sciences. 4th ed. Beijing: CRC Press.
Bisswanger, H., 2013. Practical Enzymology. 5th ed. Oxford: John Wiley & Sons.
Blankenship, L., 2012. Colonization Control of Human Bacterial Enteropathogens in Poultry. 5th ed. Oxford: Academic Press.
Copeland, R.A., 2013. Methods for Protein Analysis: A Practical Guide for Laboratory Protocols. 6th ed. Kansas: Springer Science & Business Media.
David W. Burden, D.B.W., 2012. Biotechnology Proteins to PCR: A Course in Strategies and Lab Techniques. 5th ed. London: Springer Science & Business Media.
Donglu Zhang, S.S., 2012. ADME-Enabling Technologies in Drug Design and Development. 3rd ed. Beijing: John Wiley & Sons.
Freshney, R.I., 2011. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. 6th ed. Salt Lake: John Wiley & Sons.
Ganapathy-Kanniappan, S., 2018. Advances in GAPDH Protein Analysis: A Functional and Biochemical Approach. 3rd ed. Chicago: Springer.
Hock, F.J., 2015. Drug Discovery and Evaluation: Pharmacological Assays. 4th ed. London: Springer International Publishing.
Ninfa, A.J., 2009. Fundamental Laboratory Approaches for Biochemistry and Biotechnology. 5th ed. London: Wiley.
Rosenberg, I.M., 2013. Protein Analysis and Purification: Benchtop Techniques. 2nd ed. New York: Springer Science & Business Media.
Stephenson, F.H., 2010. Calculations for Molecular Biology and Biotechnology: A Guide to Mathematics in the Laboratory. 2nd ed. New York: Academic Press.
Thomas E. Crowley, J.K., 2014. Experiments in the Purification and Characterization of Enzymes: A Laboratory Manual. 3rd ed. New York: Academic Press.
William S. Adney, J.D.M.J.R.M.K.T.K., 2009. Biotechnology for Fuels and Chemicals: The Twenty-Ninth Symposium. 4th ed. Cambrige: Springer Science & Business Media.
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