Lab Report PDF

Preview

Summary

Determining Protein Concentration Of Two Unknown Substances Using The Bradford Assay
...

Description

Determining Protein Concentration Of Two Unknown Substances Using The Bradford Assay

INTRODUCTION The Biuret test is done to indicate if protein is present but it does not inform one on how much protein is present. For that, the Bradford assay must be done to determine the total protein concentration. This assay uses a dye, Coomassie Brilliant Blue G-250, which was first reported by Dr. Marion Bradford in 1976. The dye exists in it cationic state and takes on a reddish-brown color (peak absorption of the dye in this state). The dye turns blue and absorption shifts from 470 nm to 595 nm when in contact with amino acids in proteins. The more protein present in the substance will result in a darker shade of blue. This assay allows the results to be measured qualitatively by eye, or quantitatively with a spectrophotometer. A spectrophotometer helps measure the amount of light that a sample can absorb. According to Beer’s Law, this absorbance is related to the concentration of the chemical compound being analyzed. This relationship is linear and holds true for absorbance values between 0.1 and 1.0. The spectrophotometer that we used was the Genesys 30, which consists of a light source, a sample holder, and a detector. The objective of this experiment is to determine the concentration of protein in the two “unknown” substances.

MATERIALS AND METHODS

The materials needed for the standard curve were: GENESYS 30 Spectrophotometer, 1 x Bradford dye reagent (containing Coomassie blue), set of seven bovine y-globulin (BGG) standards: 0.125, 0.250, 0.500, 0.750, 1.000, 1.500 and 2.000 mg/ml in microtubes, 1 microtube

to mix the Blank, 8 microcuvette, 1 x phosphate buffered saline (PBS) solution, 100-1000 microliter digital pipette and tips, 10-100 microliter digital pipette and tips, indelible glass/plastic marking pen

The following procedure was done to determine the standard curve. The tube containing the Bradford dye reagent was gently inverted to mix it. Then, Bradford dye reagent was added to each microtube containing 20 uL of a known concentration of bovine y-globulin (BGG) and to a clean, empty microtube labeled "blank". 20 uL of 1 x PBS was then added to microtube labelled "blank" then was incubated at room temperature for 5-10 mins. While waiting for the incubation period to elapse, microcuvettes were labelled (B, 1, 2, 3, 4, 5, 6, 7) making sure to only hold them on the rigid sides. At the end of the incubation period, the entire contents of each microtube was poured to the appropriately labeled microcuvette. Then GENESYS 30 was plugged in, set up at a wavelength of 595 nm and Mode of ABS. Then we pressed 0.00. opened the lid, and placed the microcuvette containing the blank in the chamber. We gently closed the lid; pressed the 0.00 key to set the blank; open the lid; remove the blank.

After setting the blank, it was time to measure the sample. We inserted microcuvette containing a standard sample in chamber, gently closed the lid, and recorded the absorbance value. After, we opened the lid and removed the standard. The same steps were repeated for the remaining standards and the data was recorded in the results section.

After determining the standard curve, we determine the protein concentration in two different types of milk. The materials needed for this procedure were: GENESYS 30

Spectrophotometer, 1 x Bradford dye reagent (containing Coomassie blue), two milk samples, microtubes, microcuvettes, blank microcuvette from standard curve procedure,1 x phosphate buffered saline (PBS) solution, 100-1000 ul digital pipette and tips, 10-100 ul digital pipette and tips, indelible glass/ plastic marking pen.

The following procedure was done to determine the protein concentration. First a 1:10 dilution of each unknown sample was prepared in PBS as follows: two clean microtubes was labeled - A 1:10 and B 1:10; A for cow milk and B for almond milk. The 100-1000 uL digital pipette was used deliver 900 uL of 1 x PBS into each of the microtubes. Then he 100-1000 uL digital pipette and a fresh tip was used for each unknown sample to deliver 100 uL of the appropriate sample to the correctly labeled microtube. Afterwards, microtubes were shut and gently inverted five to ten times to mix the dilution. We further diluted each of our 1:10 unknowns as follows: A. Label clean microtubes as 1:20, 1:40, and 1:80 for each sample: A and B B. Using the 100-1000 uL digital pipette, deliver 500 uL of 1 x PBS to each microtube. C. Use a fresh tip to transfer 500 uL of the 1:10 dilution of unknown A to the 1:20 tube for sample A. Cap the tube tightly and invert the tube five to ten times to mix the dilution. D. Use a fresh tip to transfer 500 uL of the 1:20 dilution of unknown A to the 1:40 tube for sample A. Cap the tub tightly and invert the tube five to ten times to mix the dilution.

E. Use a fresh tip to transfer 500 uL of the 1:40 dilution of unknown A to the 1:80 tube for sample A. Cap the tub tightly and invert the tube five to ten times to mix the dilution. The previous steps were repeated for unknown sample B. Microtubes were then labelled for the final aliqouts of the 1:20, 1:40, 1:80 dilutions of unknown samples A and B. Using the 100-1000 uL digital pipette, we delivered 1000 uL of Bradford dye reagent to each microtube. With the 10-100 uL digital pipette and using a fresh tip for each of our diluted milk samples, we pipetted 20 uL of each dilution into the correctly labeled microtube. We tightly closed each of the microtubes, gently inverted each microtubes three times to mix the reagents and incubated at room temperature for 5-10 minutes. While waiting for the incubation to elapse, we used a permanent marker to label 6 microcuvettes (A 1:20, A 1:40, A 1:80, B 1:20, B 1:40 and B 1:80) and made sure to hold the microcuvettes on the ridged sides only, and to place your labels at the top of the ridged side of the microcuvettes. We poured the microtube contents into appropriately labelled microcuvettes. While our samples were incubating, we made sure the spectrophotometer is plugged in, turned on (to warm up) and the wavelength is set to 595 nm, and set to read absorbance. We pressed 0.00, opened the lid, placed the microcuvette containing the blank in chamber, gently closed the lid; pressed the 0.00 key to set the blank; opened the lid and removed the blank. We then inserted the microcuvette containing the sample in chamber, gently closed the lid; read and recorded the absorbance value; opened the lid and removed the sample. The same steps were repeated for the remaining samples and we record our data as indicated in the Results section.

RESULT

Table 1: The standard Curve Data Cuvett e 1 2 3 4 5 6 7

Standard Concentration (mg/ml) 0.125 0.250 0.500 0.750 1.000 1.500 2.000

Absorbance 0.091 0.197 0.411 0.639 0.756 0.786 1.297

Graph 1: Standard Curve

Standard Curve Absorbance

1.5 1

f(x) = 0.65 x

0.5 0 0.000

0.500

1.000

1.500

2.000

2.500

Standard concentration (mg/ml)

Table 2: Unknown Analysis Unknown A:

Dilution (1:20) (1:40) (1:80) Unknown B:

Absorbance 0.863 0.502 0.28

Dilution

(1:20)

Absorbance 0.241

Dilution

Original

Published

Concentration

Concentration

Protein

(mg/ml) 1.33 0.77 0.42

(mg/ml) 26.6 30.8 33.6

Concentration

Dilution

Original

Published

Concentration

Concentration

Protein

(mg/ml) 0.37

(mg/ml) 7.4

Concentration

(1:40)

0.093

0.14

5.6

DISCUSSION From table 1, the standard curve was able to be drawn. The independent variable is placed on the y-axis and that is the absorbance of the substance. The dependent variable is placed on the x-axis and that is the standard concentration of the protein. The dependent variable is the concentration because that is the measurable changes that occur due to manipulating the independent variable. The independent is the absorbance because that is what is being tested. When the regression line is drawn, we can see that the line does not go through all the points because the data point is not perfectly exact, as the spectrophotometer flashes different numbers of absorbance before being steady. From the spectrophotometer we got the absorbance for each sample and from the standard curve we were able to find the standard concentration. To find the dilution concentration we used y = 0.6479x For Unknown A dilution 1:20 we did: 0.863 = 0.6479 x x = 0.863 / 0.6479 The same calculation was done for each dilution.

To find the original concentration for each dilution, we had to multiply each dilution concentration by its ratio. For example, for the dilution 1:20 for unknown A, 1.33 was multiplied by 20 and for the 1:40, 0.77 was multiplied by 40. The same was done for all the other dilutions.

In order to find the published protein concentration, the US Department of Agriculture Nutrition Database was used and then they were compared. CONCLUSION Through the experiment, we were able to use the linear regression method of the two unknown substances at different dilution ratio and by plotting the data from concentration and absorbance to get the standard curve. Using the standard curve, we determined the concentration of protein of the cow milk and almond milk at different dilution ratio

LITERATURE CITED Principles of Biology I Lab Manual, Nassau Community College Bradford, MMB. (1976). A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254 US Department of Agriculture Nutrition Database, Full Report (All Nutrients): 45204905, COWS MILK, UPC: 763528306046 Retrieved

from:

https://ndb.nal.usda.gov/ndb/foods/show/45204905?

fgcd=&manu=&format=&count=&max=25&offset=&sort=default&order=asc&qloo kup=cow+milk&ds=&qt=&qp=&qa=&qn=&q=&ing = US Department of Agriculture Nutrition Database,

Full Report (All

Nutrients): 45135768, ALMOND MILK, UPC: 744473909544 Retrieved

from:

https://ndb.nal.usda.gov/ndb/foods/show/45135768?

fgcd=&manu=&format=&count=&max=25&offset=&sort=default&order=asc&qlookup=almon d+milk&ds=&qt=&qp=&qa=&qn=&q=&ing=...