Protein concentration determination using Bradford assay

2A-PH, Group 2, Ballada, T., Banares, S., Belen, R., Bergonia R., Caraig, R.Z., and Cana, N.Department of Pharmacy, Faculty of Pharmacy, University of Santo Tomas,

España Boulevard, 1015 Manila, Philippines

ABSTRACT

Identification of a substance by its protein concentration can be done by spectrophotometry and Bradford assay, which includes combining Coomassie G-250 dye with protein solutions. Six test tubes were prepared with different volume of standard and water. After subjecting to a spectrophotometer, the absorbance of each sample was determined. The dye used changed to darker colors when in contact with a higher protein concentration, a higher absorbance meant a higher concentration. Correlation between absorbance and concentration is determined, R2 is equal to 0.9721 Also, the line of best fit was calculated, y = 0.0153x + 0.0052.

INTRODUCTION

The Bradford assay is a fast and fairly accurate method for the estimation of protein concentration.

The method is based on the proportional binding of the dye Coomassie to proteins. The more protein present, the more Coomassie binds. Furthermore, the assay is colorimetric, as the protein concentration increases, the color of the test sample becomes darker. Coomassie absorbs at 595 nm. The protein concentration of a test sample is determined by comparison to that of a series of protein standards known to reproducibly exhibit a linear absorbance profile in this assay. [1]

Figure 1. Structure of Coomassie Brillian Blue G-250

To calculate absorbances of the standards and the unknown samples, spectrophotometry is used. A spectrophotometer consists of a spectrometer to produce light of a specific color and a photometer to measure the intensity of the light [2]. Therefore, if a container (or more formally, a cuvette) of liquid of some opacity is placed between the spectrometer and the photometer, the photometer reader would change depending on the amount of light able to pass through the cuvette and the absorbance level could be recorded.

METHODOLOGY

A series of test tube were prepared as follows:

1 2 3 4 5 6

mL standard

0 0.2 0.4 0.6 0.8 1.0

mL H2O 1.5 1.3 1.1 0.9 0.7 0.5

Table 1. Different volumes of standard and voumes of H2O in different test tubes

The BSA standard was diluted to 10 mcg/mL. Then, 1.5 mL of Bradford reagent was added to the individual test tube and was allowed to stand for 5 minutes. The samples were transferred into individual cuvettes and were inserted to the spectrophotometer. At, 595 nm, the absorbance was read. The data was gathered.

RESULTS AND DISCUSSIONS

The results in Table 2 shows the data gathered in Bradford assay after undergoing the UV-vis spectrophotometer.

Table 2. Concentration and Absorbance using UV-vis Spectrophotometer

The concentration seen in Table 2 was determined using the formula C1V1 =

C2V2 where C is the concentration and V is the volume.

0 1 2 3 4 5 6 7 80

0.020.040.060.08

0.10.12

Concentration vs. Absorbance

Figure 3. Graph showing the absorbance and concentration of the samples

After finding the absorbances of the

standards shown in table 2, we plotted the

concentrations of the standards versus their

absorbances shown in figure 3. The R2 value

of 0.9721 indicated a strong correlation

between the concentration and the

absorbance. We use this data to calculate the

protein concentrations of the samples. By

using computer software, we were also able

to calculate the equation of the line of best

fit which is y = 0.0153x + 0.0052.

CONCLUSION

The higher the concentration of

BSA, the greater is the absorbance. The

result is more reliable if the correlation

between concentration and absorbance is

greater. Figure 3 shows that the curve line

formed by the results is close in structure to

mL BSA

[BSA] ug/mL ABS

0.0 0.000 0.0000.2 1.33 0.0320.4 2.67 0.0520.6 4.00 0.0610.8 5.33 0.0801.0 6.67 0.112

the drawn straight line which tells us that the

result is close to the expected output.

REFERENCES

Crisostomo, A. & et. al. (2010). Laboratoy General Biochemistry. Quezon City: C & E Publishing, Inc.

Ernst, O. (2012). Bradford protein assay. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164080/

Olson, B. (2013). "Bradford Assay. UNL classroom server Biochemistry protocols." http://wwwclass.unl.edu/biochem/protein_assay/bradford_assay.htm