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 (100μg/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 A595 against concentration (μg/mL). Also, this was used to determine the concentration of the unknown sample, which is 19.8μg/mL.

INTRODUCTION

The objective of the experiment is to quantitatively determine protein concentration in a given sample though Bradford assay.

EXPERIMENTAL

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 4mL

standard

0 0.10 0.15 0.20

mL H2O 1.50 1.40 1.35 1.305 6 7 8 9

0.25 0.30 0.35 0.40 0.451.25 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 (μg/mL)

RESULTS AND DISCUSSION

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.

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 ultraviolet-visible 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.

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:

mL standard× 100 μgmL

=x

xtotalmL∈eachtubewhich (3.0mL)

=concentration

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× 100μgmL

=10 μgmL

÷3.0mL=3.33 μg/mL

0.15× 100μgmL

=15 μgmL

÷3.0mL=5 μg/mL

0.20× 100μgmL

=20 μgmL

÷3.0mL=6.67 μg/mL

0.25× 100μgmL

=25 μgmL

÷3.0mL=8.33 μg/mL

0.30× 100μgmL

=30 μgmL

÷3.0mL=10 μg/mL

0.35× 100μgmL

=35 μgmL

÷3.0mL=11.67 μg /mL

0.40× 100μgmL

=40μgmL

÷3.0mL=13.33μg /mL

0.45× 100μgmL

=45μgmL

÷3.0mL=15 μg /mL

Table 2 Concentrations of Test Tubes

Test Tube No. Concentration(μg/mL)

1 02 3.333 54 6.675 8.336 107 11.678 13.339 1510 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. A5951 02 0.0973 0.1914 0.1335 0.2926 0.3567 0.3648 0.3759 0.377

10 0.622

Table 4 Albumin Standard Curve Data

Test Tube No.

Concentration(μg/mL)

A595

1 0 02 3.33 0.0973 5 0.1914 6.67 0.1335 8.33 0.2926 10 0.3567 11.67 0.3648 13.33 0.3759 15 0.377

10 x 0.622Table 4 is just a combined data of table 2

and 3. Also with the data of table 4, it will result to figure 3.

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 two equations which are y=0.0521 xand y=0.056 x−0.0271. The latter equation is to be

used because 0.056 x is closer to 1. x 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+by−bm

=mxm

x= y−bm

The solution to solve for the concentration of the unknown sample is

y=0.056 x−0.0271

x= y−bm

x=0.622−(−0.0104)0.0318

x=19.89 μg/mLIf the concentration of the unknown

sample is to be plotted, the albumin standard curve will look like figure 4

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