QUANTITATIVE ANALYSIS OF PROTEIN Gabrielli Anne B. Saavedra, Juan Miguel C. Santos, Rea Christine B. Tapel, Kyra Zara N. Tieng, Anthea Jae O. Ungsod, Jacquelyn Faye S. Usero Group 7 2G Pharmacy Pharmaceutical Biochemistry Laboratory
ABSTRACT A series of test tubes were prepared in the experiment with the following reagents: Bovine serum albumin (BSA) standard (100 μ g/mL), Bradford reagents, distilled water and an unknown sample. The first tube contained distilled water only and the tenth test tube contained the unknown sample only. The remaining test tubes were a combination of distilled water and bovine serum albumin (BSA) standard that would total to 1.5mL of solution. 1.5mL of the Bradford reagent was added to all of the test tubes. The absorbance at 595nm was read and the data retrieved was used to construct the albumin standard curve by plotting A 595 against concentration ( μ g/mL). Also, this was used to determine the concentration of the unknown sample, which is 19.8 μg/mL .
INTRODUCTION The objective of the experiment is to quantitatively determine protein concentration in a given sample though Bradford assay.
EXPERIMENTAL
The Bradford assay is commonly used to determine the total protein concentration of a sample. [1] The initial color of the solutions of the test tubes was red. Upon the addition of the Bradford reagent, the solution turned blue which indicates that the solution is acidic and its absorbance it at 595nm. The results can be seen in figure 1.
A. Compounds tested (or samples used) Bradford reagent, Bovine serum albumin (BSA) standard, distilled water and unknown sample.
B. Procedure A series of test tubes was prepared as follows: Table 1 Preparation of Test Tubes
Tube # 1 2 3 4 mL 0 0.10 0.15 0.20 standar d mL H2O 1.50 1.40 1.35 1.30 5 6 7 8 9 0.25 0.30 0.35 0.40 0.45 1.25 1.20 1.15 1.10 1.05 1.5 mL of unknown sample was placed in a test tube, which was labeled as 10. 1.5 mL of Bradford reagent was added to each test tube. The test tubes were mixed well and left to stand for 5 minutes. 3 drops from each test tube were placed in a microplate. The samples on the microplate were placed in a UV-Vis Spectrophotometer. The data that was retrieved was used to construct the albumin standard curve by plotting A595 against concentration ( μ g/mL)
RESULTS AND DISCUSSION
Figure 1 Test Tube
Ultraviolet-visible spectrophotometry is the absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. The microplate containing few drops of each of the test tubes underwent ultravioletvisible spectroscopy at absorbance 595nm. The instrument used was an ultraviolet-visible spectrophotometer. The data retrieved in figure 2 is row B, which is used to construct the albumin standard curve.
Table 2 Concentrations of Test Tubes
Test Tube No.
Figure 2 Data from UV-Vis Spectrophotometer
In constructing the albumin standard curve, the A595 is plotted against concentration ( μ g/mL). The concentration of each test tube must be calculated by using this formula:
100 μg =x mL x =concentration total mL∈each tube which (3.0 mL)
mL standard ×
The first test tube will have no concentration since there was no mL standard added to it. The following are the solutions for the test tubes 2 to 9 respectively below. As for the concentration of the tenth test tube or the unknown sample, it is solved using the albumin standard curve.
0.10 ×
100 μg 10 μg = ÷ 3.0 mL=3.33 μg /mL mL mL
0.15 ×
100 μg 15 μg = ÷ 3.0 mL=5 μg /mL mL mL
0.20 ×
100 μg 20 μg = ÷ 3.0 mL=6.67 μg/mL mL mL
0.25 ×
100 μg 25 μg = ÷ 3.0 mL=8.33 μg/mL mL mL
0.30 ×
100 μg 30 μg = ÷ 3.0 mL=10 μg /mL mL mL
0.35 ×
100 μg 35 μg = ÷ 3.0 mL=11.67 μg/mL mL mL
0.40 ×
100 μg 40 μg = ÷3.0 mL=13.33 μg/mL mL mL
0.45 ×
100 μg 45 μg = ÷3.0 mL=15 μg/mL mL mL
Concentration ( μ g/mL) 0 3.33 5 6.67 8.33 10 11.67 13.33 15
1 2 3 4 5 6 7 8 9 10 x Before the albumin standard curve can be made, the A595 of test tubes 2 to 10 are subtracted to the A595 of test tube 1. The reason is test tube 1’s absorbance should be zero. This will result to the data shown in Table 2. Table 3 Absorbance at 595nm
Test Tube No. 1
A595
2 3 4 5 6 7 8 9 10
0.097 0.191 0.133 0.292 0.356 0.364 0.375 0.377 0.622
0
Table 4 Albumin Standard Curve Data
Test Tube No. 1
Concentration ( μ g/mL)
A595
0
2 3 4 5 6 7 8 9 10
3.33 5 6.67 8.33 10 11.67 13.33 15
0 0.097 0.191 0.133 0.292 0.356 0.364 0.375 0.377 0.622
x
Table 4 is just a combined data of table 2 and 3. Also with the data of table 4, it will result to figure 3.
y=0.056 x−0.0271 y−b x= m 0.622−(−0.0104) x= 0.0318 x=19.89 μg /mL If the concentration of the unknown sample is to be plotted, the albumin standard curve will look like figure 4
Figure 3 Albumin Standard Curve based on Table 4
For the concentration of the unknown sample, the slope of the albumin standard curve (figure 3) must be used. The formula for this is y=mx +b . It is shown in figure 3 that there are y=0.0521 x and two equations which are y=0.056 x−0.0271 . The latter equation is to x be used because 0.056 x is closer to 1. is to be solved in the formula because concentration is plotted at the x-axis which is the unknown. The derived formula is below:
y=mx +b y −b mx = m m y−b x= m The solution to solve for the concentration of the unknown sample is
Figure 4 Final Albumin Standard Curve
Based on figure 4, the unknown sample did not meet the range of absorbance based on the data and the plot graph. This can be due to inaccuracy in pipetting and contamination of the reagents used. To be able to meet the range, the amount of distilled water and the standard should be adjusted to a certain concentration.
REFERENCES [1] Crisostomo, Angelica C., et al (2010). Laboratory Manual in General Biochemistry. Quezon City: C & E Publishing House. Pg. 25-26
