Bradford Formal Report

The Isolation of Myoglobin from Minced Beef by Salt-Induced Precipitation and the Determination of the Protein Concentration of Myoglobin from Minced Beef Using the Bradford Assay Chua, K.M.D., Cocjin, C.M.PH.R, Dizon, J.A.H., Donato, L.P.G., Dumaplin, R.A.L, Francisco, R.A.2B-PH, Group 3, Department of Pharmacy, Faculty of Pharmacy, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines ABSTRACT The protein, myoglobin, is found in muscle cells of animals [1].The Bradford assay is a common method used to determine the protein concentration of a given sample [2]. This experiment was conducted to successfully isolate the protein myoglobin from minced beef and to quantitatively determine the protein concentration of myoglobin that was extracted from minced beef by saltinduced precipitation. The Bradford method is based on the binding of acidic Coomasie dye, a coloring reagent, to proteins in which protein concentration is directly proportional to the dye [3]. The standard protein that was used in this experiment is Bovine Serum Albumin (BSA) which the Bradford assay is sensitive to than most common proteins [4]. Seven analytes were prepared using assigned volumes of distilled water and Bovine Serum Albumin while one test tube was left blank. The UV-Vis Spectrophotometer was used to measure the absorption of the analytes. Apart from the nine test tubes that were prepared, another was prepared and it contained the unknown protein. Since known concentrations of protein were prepared, the unknown protein can be estimated and can be compared to their absorbance. To compute for the protein concentration, the linear regression equation was used. To graphically represent the relation of concentration and absorption, a Standard Calibration Curve was devised. It was then used to deduce the protein concentration of the unknown sample. INTRODUCTION Proteins are large, complex molecule that are composed of numerous chains of amino acids in a specific order [5]. There are 20 amino acids that are commonly found in proteins [2]. They are required for the structure, function, and regulation of the cells, tissues, and organs of the body [5]. They are classified according to their functions. A few examples of proteins are actin, immunoglobulin, and myoglobin [6]. Myoglobin is a protein that can be found in the muscle cells of animals. It functions as an oxygen-storage unit that provides oxygen to working muscles [1]. Proteins can be isolated from a crude sample. The most common way to

precipitate proteins is through salt-induced precipitation [7]. At low concentrations of salt, the solubility of the protein more often than not increases slightly [7]. When this happens, it is called salting in. An assay is used to measure the concentration of a substance; therefore, a protein assay measures the concentration of proteins [8]. A rapid and accurate method to estimate protein concentration is essential in protein studies [9]. The Coomassie blue method otherwise known as the Bradford method was developed by Marion M. Bradford in 1976 [8]. The Bradford assay relies on the binding of the dye, Coomassie

blue G-250, to protein [9]. Coomasie blue G-250 binds to protein given that it is in an acidic solution [8]. Considering that the Bradford assay is colorimetric, it can be assumed that as the protein concentration increases, the darker the color of dye becomes. The Coomassie dye has a maximum absorbance of 590nm [9]. Hence, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form [9]. This is frequently

achieved by measuring the absorbance of the solution at 595 nm [9].

METHODOLOGY

transferred into a new beaker using filter paper. The extract was divided and put in separate centrifuge tubes. The individual test tubes containing the extract were centrifuged at 13,000 x g for five minutes. After being centrifuged, 1.5 mL of the supernatant was transferred to another beaker. Approximately, 0.30-0.35 g of ammonium sulfate [(NH4)2SO4] crystals were ground into a fine powder and was added to the supernatant. It was mixed gently until the powder was entirely dissolved in the supernatant. Frothing was avoided. The sample was again centrifuged at 13,000 x g for five minutes. The supernatant was then decanted, leaving only myoglobin. The myoglobin was set aside.

For the isolation of myoglobin, the apparatus that were used were a centrifuge, a graduated cylinder, a triple beam balance, a watch glass, a stirring rod, Pasteur pipettes, beakers, a funnel, filter paper, and centrifuge tubes. The reagents and materials that were used were minced beef, ammonium sulfate [(NH4)2SO4] crystals, 70% buffer-diluted ammonium sulfate [(NH4)2SO4] solution with a pH of 7.5. For the protein assay, the apparatus that were used were, test tubes, a test tube rack, pipette, rubber aspirator, tape, a stirring rod, and a UV-Vis Spectrophotometer. The reagents that were used for the assay were, the Bradford reagent, Bovine serum albumin standard (BSA), and the evaporated milk sample. Six grams of minced beef was weighed on a triple beam balance with the aid of a preweighed watch glass. Six milliliters of the 70% ammonium sulfate [(NH4)2SO4] solution was measured using a graduated cylinder. The minced beef and the 70% ammonium sulfate solution were placed together in a beaker. The mixture was stirred for one minute to allow the release of myoglobin. The dark-red extract was

The objectives of this experiment are: (1) to isolate myoglobin from minced beef by saltinduced precipitation and (2) to quantitatively determine the protein concentration of a given sample through Bradford assay.

After the reagents and materials for the Braford assay were prepared, a series of test tubes were prepared with containing the following: Tube No.

1

2

3

4

5

6

7

8

9

10

mL distilled H2O mL Standar d BSA

2. 5

2.0

2.0

1.5

1.5

1.0

1.0

0.5

0.5

2. 0

0

0.5

0.5

1.0

1.0

1.5

1.5

2.0

2.0

0. 5

There were four standards that were prepared by the class. Two groups were assigned per standard. The first test tube was left blank; while the last test tube contained the unknown sample. Subsequently, 2.5 mL of Bradford reagent was added to all 10 test tubes. The mixtures were mixed using a stirring rod and they were placed in a test tube rack to stand for 5 minutes. The absorbance was read at 595 nm within the hour. The albumin standard curve was constructed by plotting the A595 against the concentration (mg/mL). The linear regression was also used to compute for the concentration. RESULTS AND DISCUSSION Myoglobin is an oxygen-carrying and storage protein of muscle that resembles hemoglobin but contains only one subunit and one heme as part of the molecule (rather than the four of hemoglobin), and with a molecular weight approximately one quarter than that of hemoglobin [10]. The protein, myoglobin, can be extracted from the minced beef using the method called salt-induced precipitation. Saltinduced precipitation is the most common way to precipitate proteins [7]. Due to the bad quality of the beef that was used in the experiment, the group failed to extract myoglobin from the beef. This was proven using the different qualitative color reactions. Despite this, the group still performed all the tests on the sample. The Bradford assay is a fast, convenient, and efficient way of determining the amount of protein present in a solution [4]. The Coomasie dye that is present in the Bradford reagent shifts from 465 nm to 595 nm when

protein is bound to the dye [4]. The more protein is present in a solution, the darker the dye got. A UV-Vis Spectrophotometer is used to measure the how much light was absorbed by the samples. Two trials were done and the average of the two values were the ones that were used in the graph. However, time was scarce and the class was not able to use the UV-Vis Spectrophotometer. Instead, the instructor provided the class the data. The data is tabulated as seen below. TABLE 1. DATA COLLECTED PROVIDED Sample

Total protein concentration (mg/mL)

A595 Trial 1

A595 Trial 2

A595 Average

Blank

0

0

0

0

Standard 1 Standard 2 Standard 3 Standard 4 Unknow n

0.050

0.2485

0.2265

0.2375

0.040

0.2245

0.2036

0.2141

0.030

0.1864

0.1910

0.1887

0.020

0.1529

0.1512

0.1521

0.1421

The data above will be used to make the standard curve graph with protein concentration as the x-axis and the A595 as the y-axis. Standard Curve Graph 0.4 A595 0.2 0

0

0.01 0.02 0.03 0.04 0.05 0.06 Protein Concentration

To get the A595 of the unknown protein, the linear regression equation was used. The formula is seen in the figure below. FIGURE 1: LINEAR REGRESSION EQUATION

Linear regression ( y )=a+ bx

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lmgren/Glossary/Definitions/DefP/protein.html [6] What are proteins and what do they do? (2016). Retrieved March 27, 2016 from https://ghr.nlm.nih.gov/handbook/howgenes work/protein [7] Salt-Induced Precipitation of Proteins (n.d.). Retrieved March 27, 2016 from http://www.rpi.edu/dept/chem-eng/BiotechEnviron/PRECIP/precpsalt.html

[8] Protein Analysis-Determination of Protein Concentration. (2011). Retrieved March 27, 2016 from http://public.callutheran.edu/~revie/biochem istry/Protein-analysis-lab.pdf

[3] Bradford Concentration Protein Assay. (2001). Retrieved March 27, 2016 from https://ww2.chemistry.gatech.edu/~lw26/bC ourse_Information/4581/techniques/bradford /bradford.html

[9] Kruger, N. (n.d.). Bradford Method for Protein Quantitation. Retrieved March 27, 2016 from http://cst.ur.ac.rw/library/Food %20Science%20books/batch1/Food %20Science%20and%20Technology %20Published%20articles/Analysis %20Protocols/The%20bradford%20method %20for%20protei%20quantitation.pdf

[4] Caprette, D. (1995). Bradford protein assay. Retrieved March 27, 2016 from http://www.ruf.rice.edu/~bioslabs/methods/p rotein/bradford.html

[10] Myoglobin. (n.d.) Farlex Partner Medical Dictionary. (2012). Retrieved March 28 2016 from http://medicaldictionary.thefreedictionary.com/myoglobin

[5] Lefers M. (2004). Protein. Retriever March 27, 2016 from http://groups.molbiosci.northwestern.edu/ho