QUANTITATIVE DETERMINATION OF THE TOTAL PROTEIN CONCENTRATION USING THE BRADFORD METHOD

Audrey Coleen Declaro, Anne Marie Garcia, Kester Immer Guballa, Elizann Faye Hipolito, and Manjoric Jardiolin Group 3 2G Medical Technology Biochemistry Laboratory

ABSTRACT
For this experiment, eleven bottles were prepared; one test tube was left blank, while the other eight are filled with the designated volume of Bovine Serum Albumin (BSA), the standard solution, and water. The tenth test tube was filled with 1.5 ml of the standard solution, undiluted, while the eleventh test tube was filled with 1 ml of the standard and 4 ml of water. After the preparation, 1.5 ml of the Bradford reagent was added to each test tube. The contents were mixed well and were left to stand for about 5 minutes. The test tubes were then subjected to UV-VIS Spectrophotometry. The readings from the spectrophotometer were gathered by the assigned member of the group. After gathering the necessary data, a standard curve was plotted and the concentration of the sample solution was determined with the aid of the graphical method.

INTRODUCTION
Protein quantity determination is necessary before processing protein samples for isolation, separation and analysis by chromatographic, electrophoretic and immunochemical techniques. The establishment of standard curves is a concept based on a direct relationship between two parameters such that if you can determine for certain one part of the related association, you can derive the other. This requires that you have one parameter that is precise, definable, and measurable. This utility is extended to the biological and chemical laboratory as a tool used to determine an unknown concentration of substance in a particular volume (suspension) of liquid. By measuring the fixed and known concentrations of a substance, you may derive another physical component of that suspension whether it be radiological, fluorescent, luminescent, or other. In a more focused application, the optical density is the derived physical descriptor that is dependent on the concentration of protein in suspension. [1] Bradford method is a common colorimetric method to determine protein concentration in a sample solution. The Bradford method of protein determination is based on the binding of a dye, Coomasie Blue G, to the protein. The Bradford assay is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than the assays described above. Like the other assays, however, its response is prone to influence from non protein sources, particularly detergents, and becomes

progressively more nonlinear at the high end of its useful protein concentration range. The response is also protein dependent, and varies with the composition of the protein.

EXPERIMENTAL PROCEDURE
A. Samples/Reagents used Bovine Serum Albumin (BSA) Unknown protein, Bradford reagent standard,

B. Procedure A series of test tubes were prepared by adding the standard, the Bovine Serum Albumin, and the corresponding amount of water in eight of the test tubes. One was left blank. The mixture of the two must be equal to 1.5 ml. In another set of test tubes, one test tube was filled with the unknown, undiluted sample, and another test tube was filled with 1 ml of the unknown protein and 4 ml of water. 1.5 ml of the Bradford reagent was added to each test tubes and the test tubes were allowed to stand for five minutes after mixing. The prepared solutions were then subjected to UV-VIS spectrophotometry for the measurement of the absorbance. The solutions were transferred into their corresponding cuvettes and the values were written down. After getting the data from the spectrophotometer, the standard curve was constructed by plotting the A540 against the concentration of the protein.

Results and Discussions

The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assesing protein concentrations. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range. The spectrophotometer was used to determine the absorbance of each solution, (Table 1). This instrument measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I/Io is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance: A= log (%T/100%)
Table 1. Absorbance (A) measured using UV-Vis Spectrophotometer Test tube 1 2 3 4 5 6 7 8 9 10 11 Absorbance, 540 nm 0.59 A

solution (1.5 mL). The computed results are shown in Table 2. The concentration of the unknown was represented with X.
Table 2. Computed concentration of the standard solutions Test tube 1 2 3 4 5 6 7 8 9 C2 0

6.667
10.000 13.333 16.667 20.000 23.333 26.667 30.000

The concentration of the unknown protein was determined first by graphing the computed amount of protein concentration against absorbance and by using linear regression method. Through graphical method the concentration of the unknown protein was determined the protein concentration on the xaxis while absorbance was in the y-axis.

Protein Standard Curve

3 2.5
Absorbance

y = 0.0833x R = 0.4294

1.001 A 1.243 A 1.525 A 1.671 A 1.69 A 1.908 A 2.008 A 2.127 A 1.576 A 1.049 A

2 1.5 1 0.5 0 0 20 40
Protein Concentration mg/mL

Absorbance, 540 nm Linear (Absorbance , 540 nm)

Figure 1. Protein Standard Curve Chart

The protein concentration of each standard was determined by using the dilution equation, where C1 is the concentration of Bovine Serum Albumin (BSA) V1 is the volume of standard protein, C2 is the protein concentration determined in this equation and V2 is the total volume of the

The best-fit line was drawn for the graph was not linear due to several errors and factors that affected the experiments. For the unknown protein, the concentration was determined by tracing the point in which the absorbance, 1.280, met the best-fit line of the graph. On the other

hand, for a more accurate result the linear regression method was used to determine the protein concentration. The protein concentration of the undiluted solution is 18.92 mg/mL and the concentration of the 1:5 diluted protein is 12.59 mg/mL

REFERENCES
Bradford Assay Background. Retrieved from http://www.histosoft.com/services/background/ ba/baback.html Bradford Assay for Protein Quantification. Retrieved from http://www.oardc.ohiostate.edu/stockingerlab/Protocols/Bradford Assay.pdf Bradford Method: Colorimetric Protein Assay. Retrieved from http://bioteachnology.com/protein/bradfordmethod-colorimetric-protein-assay Bradford Protein Concentration Assay. Retrieved from http://ww2.chemistry.gatech.edu/~lw26/ bCourse_Information/4581/techniques/ bradford/bradford.html Crisostomo, A. et. al (2010). LaboratoryManual in General Biochemistry.Isolation and Characterization of Proteins. South Triangle, Quezon City: C&E Publishing, Inc.