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Protein Assay Using the Bradford Method Andres, Neil Kaila, Apacible, Fatima Anne, Ballador, Paulleen, Caguna,

April Joy, Co, Timothy Group 1 2E Medical Technology Biochemistry Laboratory ABSTRACT The Bradford Assay technique is used to determine the concentration of a given protein. It is a spectroscopic analytical procedure that is used to measure the solution's protein concentration. This method implies the use of the acidic Coomassie dye as a coloring agent, in which protein concentration is proportionate to the dye. In this experiment, UV-Vis Spectrophotometer was used to measure the absorption of the analytes, 1 blank and 7 other test tubes were assigned with a certain volume of bovine albumin standard and a volume of distilled water. The last test tube will constitute the unknown protein. The protein concentration was computed using the dilution equation. The protein concentration of the unknown was determined by using two methods: linear regression method and graphical method. INTRODUCTION binding of the dye Coomassie dye to protein, in which the dye is proportional to the concentration. Without the protein, the solution is red-brown in its acidic solution and when protein binds the pKa of the dye shifts causing it to turn blue. Thus, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form.

In many fields of protein study, an accurate and rapid method for estimation of protein concentration is essential. These determinations of proteins are quantitative in nature and can be elemental, gravimetric or spectroscopic. The Bradford assay has become the preferred method for quantifying protein in many laboratories. It is because this spectroscopic technique is simpler, faster, and more sensitive when compared with other methods. The Bradford assay also is subject to less interference by common reagents and non-protein components of biological samples. Protein assays are designed to measure the total protein in a solution; that is all of the proteins in solution. Protein assays are quantitative if the protein to be assayed is available in sufficient quantity such that one is a able to use it to create a standard curve. If this cannot be achieved, then a standard protein, such as albumin, may be used for a standard curve with the understanding that the results on the unknown protein are semiquantitative. Because most proteins are not available in large quantities, standard curves for protein assays are typically based on the use of either bovine serum albumin (BSA) or bovine gamma globulin. The Bradford assay relies on the

EXPERIMENTAL A. Compounds Tested (samples used) Bradford reagent, Bolvine serum albumin (BSA) standard (100 ug/ml), unknown protein sample B. Procedure: 9 test tubes were first prepared by filling each with varied amounts of distilled water. The first test tubes had slightly more than the latter ones, test tubes 2 and 3 for example, had a difference of approximately 0.5 ml, and the rest followed in similar fashion. We poured the BSA on 8 test tubes; not including the first, and again in various amounts, this time the preceding test tubes had more BSA than the latter ones, effectively making them less concentrated with BSA. Each test tube was then mixed thoroughly and 1.5 ml of Bradford reagent was then added into all test tubes then were left undisrupted for 5 minutes.

We then transferred the contents into 9 cuvettes, with the additional cuvette containing the sample. The prepared cuvettes were then taken downstairs to have their absorbance measured using the UV-Vis spectrophotometer. An albumin standard curve was made showing the relationship of standard mixtures. Points were connected in linear regression; in doing so, total protein concentration of sample was determined. Tube no. RESULTS AND DISCUSSION The Bradford method is a spectroscopic analytical procedure used to measure protein concentration in a certain solution. The Ultraviolet-Visible spectrophotometer is then used to measure the absorbance of the standard and unknown protein. The spectrophotometer uses light in the visible and adjacent near ultraviolet and near-infrared ranges to produce it's readings. The absorption in the visible range directly affects the perceived color of the chemicals involved. he concentration of the unknown protein was determined by two ways: first, by graphing the computed amount of protein concentration against absorbance and by using linear regression method. Through the graphical method the concentration of the unknown protein was determined, the protein concentration on the x-axis while absorbance was in the y-axis. Blank 2 3 4 5

Conc. of BSA
(ug/mL) 0 0.003 0.005 0.007 0.0083

A595 0 0.134 0.185 0.275 0.393

0.01

0.432

0.012

0.459

0.013

0.564

y = 41.976x - 0.0006 R = 0.9782 0.6 0.5 0.4 0.3 0.2 0.1 0 0 -0.1 0.002 0.004

A595

y = 41.909x R = 0.9782

0.006

0.008

0.01

0.012

0.014

REFERENCES
1. Bradford, M. M. (2000). Analyt. Biochem. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. 72,248254. 2. Bradford Method: Colorimetric Protein Assay Retrievedofromphttp://bioteachnology.com/p rotein/bradford-method-colorimetric-proteinassay 3. Crisostomo, A. et. al(2010). Laboratory Manual in General Biochemistry. Isolation and Characterization of Proteins. South Triangle, Quezon City: C&E Publishing, Inc. Ultraviolet-visible spectrophotometry. 4. Noble, J.E.; Bailey, M.J.A. (2009). Methods Enzymol. Quantitation of Protein. Retrieved from http://en.wikipedia.org/wiki/Bradford_protein _assay 5. Zuo, S.-S. and Lundahl, P. (2000). Analyt. Biochem. A micro-Bradford membrane protein assay. 284, 162164. 6. Krohn, R.I. (2001). The Colorimetric Determination of Total Protein, Current Protocols in Food Analytical Chemistry , B1.1.1-B1.1.27, John Wiley & Sons, Inc.7. Hopkins, (1993) Maton et. Al. Human Biology and Health. Englewood Cliffs, Michigan, USA: Prentice Hall 8. Kruger,N.(2003) The Handbook B.14-16, Protein Protocols