Protein Assay Using the Bradford Method

Katrina Duron, Azalea Damaris Encarnacion, Patricia Mikchaela D.L. Feliciano,Ciela Kadeshka A. Fuentes, Bea Trixia B. Gales, Ethel Princess A. Gepulango

Group 3 2F Pharmacy Biochemistry Laboratory

ABSTRACTBradford protein assay is a spectroscopic method used to determine a protein concentration in a solution; meaning, it relies on the binding of Comassie dye to protein. In this experiment, 5 test tubes were prepared which was divided per group in the class; the first test tube or the blank test tube containing 3.0 mL of distilled water while the last test tube containing 0.8 mL of bovine serum albumin (BSA) standard. 3.0 mL of the Bradford reagent was also added to each test tube. After mixing them, the test tubes were stood for 5 minutes and their absorbance was determined at 595 nm using the spectrophotometer. The absorbance of each of the test tube was determined twice and its absorbance was the average of the two trials. Finally, the concentration of the protein was determined and its absorbance (A595) was plotted against the concentration, creating the albumin standard curve. An unknown solution’s absorbance was also determined in order to compute for the linear regression.

INTRODUCTION The Bradford protein assay is a rapid and accurate method for the estimation of protein concentration. It does not require heat and also, it gives a more stable colorimetric response that is prone to influence from non-protein sources. The response becomes more non-linear at the high end of the protein concentration, protein dependent, and varies with the composition of the protein.

A spectrophotometer measures the amount of light absorbed by passing a beam, which consists of a stream of photons, through a sample. The analyte might absorb the photon when the latter encounters an analyte molecule, thus, reduces the number of photon in the beam of light as well as the light beam’s intensity.

EXPERIMENTALA. Compounds tested (or Samples used) Bradford reagent, bovine serum albumin (BSA) standard (100 µg/mL), unknown solution, distilled water.

B. Procedure 1. Absorbance of the standard protein The class prepared a series of test tubes as follows:

Table1. Standard proteinTest tube (standard)

Bovine serum albumin (BSA) standard

Distilled water

Blank 0 mL 3.00 mL1 0.20 mL 2.80 mL2 0.40 mL 2.60 mL3 0.60 mL 2.40 mL4 0.80 mL 2.20 mL

Our group was assigned to prepare standard 2 so 0.4 mL standard was diluted in a test tube with 2.6 mL distilled water.

Figure 1. Standard protein without Bradford reagent

And then, 3 mL Bradford reagent was added to the test tube. It was stood for 5 minutes.

Figure 2. Standard protein with Bradford reagent

After preparing the standard, its absorbance was gotten at 595 nm using a spectrophotometer.

Figure 3. Getting the absorbance of the standard protein

2. Standard Protein Concentration To compute for the concentration of the standard protein that was prepared, the equation C1V1=C2V2; where C1 is the concentration in 0.4 mL of the Bovine serum albumin (BSA) standard which is 1 mg/mL and C2 is the concentration of the standard protein in 3.0 mL diluted bovine serum albumin (BSA) standard which is yet to be computed.

3. Absorbance of the unknown 0.1 mL unknown was diluted using 0.9 mL distilled water.

Figure 4. Diluted unknown without Bradford reagent

0.2 mL diluted unknown was further diluted in 2.8 mL distilled water and 3.0 mL Bradford reagent was added.

Figure 5. Unknown with Bradford reagent

After preparing the unknown, it was put in a spectrophotometer to get its absorbance at 595 nm. 4. Plotting the standard curve Now that the standard protein concentration and its absorbance were already determined, the standard curve can be plotted. Let the standard protein concentration be on the x-axis while the absorbance be on the y-axis.

RESULTS AND DISCUSSION A total of 5 test tubes were prepared; the first test tube being the blank, containing only 3.0 mL distilled water. The experiment was divided within 8 groups. Group one prepared the black test tube as well as Trial 1 for standard 1 while group 2 prepared the 2nd trial for standard 1. Groups 3, our group, and 4 prepared standard 2’s 1st and 2nd trial respectively. Groups 5 and 6 were assigned in standard 3; and lastly, standard 4 was assigned to groups 7 and 8. Data were gathered from each group in the class having the results of the experiment as follows:

Table 1. ResultsAbsorbance

Test tube Standard Protein Conc.

Trial 1 Trial 2 Average

BlankStandard 1

0.067 0.033A

0.072A

0.0525A

Standard 2

0.13 0.277A

0.144A

0.2105A

Standard 3

0.2 0.381A

0.400A

0.392A

Standard 4

0.27 0.607A

0.424A

0.0155A

The standard protein concentration was computed using the equation C1V1=C2V2 as shown below:

On the other hand, the absorbance of the standard protein was determined using the spectrophotometer as shown in figure 6.

Figure 6. Absorbance of the standard protein

The standard curve can already by plotted now that the standard protein concentration and the average absorbance were already determined. Table 2 shows the standard curve, letting the concentration be on the x-axis while the absorbance be on the y-axis. The standard curve that was plotted showed a linear curve.

Table 2. Standard Curve

0 0.067 0.13 0.2 0.270

0.10.20.30.40.50.6

Standard Curve

Standard Curve

As shown in figure 7, the absorbance of the unknown was also determined in the spectrophotometer to compute for the linear regression.

Figure 7. Absorbance of the unknown To compute for the linear regression, the equation y=mx + b was used. The computation is shown below.

C1V1=C2V2

C1= 1mg/mLV2= 0.4 mL BSAC2= standard protein concentrationV2= 0.4 mL BSA + 2.6 mL distilled water (diluted BSA)

(1 mg/mL)(0.4 mL)=C2(0.4 mL + 2.6 mL)

C2= [( 1mgmL ) (0.4mL )]3.0mL

REFERENCES[1] Blauch D.N. (2009). Spectrophotometry, from

http://www.chm.davidson.edu/vce/spectrophotometry/Spectrophotometry.html 1/09/14[2] Linear Regression, from http://www.stat.yale.edu/Courses/1997-98/101/linreg.htm 1/11/14[3] Richards, S. (2012). Calculating final, or working, concentrations for journal-style scientific papers. In How to Write a Paper in Journal Style and Format, from http://abacus.bates.edu/~ganderso/biology/resources/Calculating_final_concentrations_2012.pdf 1/11/14[4] Stapel, Elizabeth. Straight-Line Equations: Slope-Intercept Form. Purplemath. from http://www.purplemath.com/modules/strtlneq.htm.  1/09/14

y=mx + by= absorbance of the unknownm= slopex= linear regressionb= intercept

The values were gotten using a scientific calculator.y= 0.733m= 0.072b=1.5560.733=0.072x + 1.556x= 8.02