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Cells and Molecules: Protein Laboratory Exercise 4 Lab Report

Winnie Mei

November 5, 2023

BIO 1205: Cells and Molecules Lab

Section 04, Tuesday 11:20 AM

Lab Partners: Anabella Yates

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Abstract

The purpose of this lab was to understand how to use the colorimetric method to find the

amount of protein in a solution. In this laboratory experiment, the researchers would use the

absorbance levels in the SpectroVis Plus to help determine the concentration of protein in the

solutions. In addition, the researchers prepared and used a standard curve to calculate the amount

of unknown protein concentration in a solution. This was done by plotting the absorbance values

and the protein concentration on a graph and finding the linear relationship between both

variables. After plotting the values on the graph, the researchers then would use the standard

curve to predict the protein concentration in unknown solutions. Additionally, the researchers

learned and understood the limitations of using the standard curve and the limitations of

colorimetric measurements. To conclude, the researchers predicted that they would be able to

detect protein concentrations in both the glycine solution and the phosphate-buffered saline

solution.
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Introduction

Proteins are macromolecules that perform many different functions such as cell

recognition, transporting molecules, and communication in the cell. In addition, proteins could

also act as enzymes, receptors, hormones, and can be structural components in the cell.

Generally, proteins consist of amino acids that are linked together in a chemical chain known as

a polypeptide. All amino acids have a carboxyl group, an amino group, and a central carbon. In

addition, each amino acid has an R-group, which is a chemical structure that is attached in

addition to the carbon. Depending on the R-group that is attached to the carbon atom, the protein

would differ in structure and function. In addition to the many functions that proteins serve, they

are also necessary for human consumption and diet. Depending on the person’s diet, proteins are

needed because the human body requires amino acids to function and make proteins. In addition,

the amount of protein needed may vary from person to person. This can be influenced by a

person’s physical activity, size and weight, metabolism, and personal health conditions.

Therefore, it is important to understand the amount of protein needed in the human body and

understand how to measure the protein levels in food.

In this lab, the protein concentration was measured in different solutions using the

“biuret” method, which detected the presence of peptide bonds by the colors in the solutions. The

researchers used this method because it can be used to assess the concentration of proteins by

measuring the amount of peptide bonds. The “biuret” method is considered a colorimetric test

because certain solutions have a specific color, which is used to determine and measure the

amount of a substance in a solution. Generally, the biuret test would display a blue color if no

protein concentration was detected in the solution, and would display a purple color if a protein

concentration was detected in the solution. In the biuret reaction, the purple color comes from the
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binding of copper ions to the amine groups created by peptide bonds. In this case, the biuret was

used to measure the amount of peptide bonds in the solution because the amino acids in proteins

are linked together through peptide bonds.

During the lab, a standard curve was used to examine the linear relationship between the

absorbance levels and the concentration of protein in the solution. The protein used in the lab is

the bovine serum albumin (BSA) which was used to calculate the standard curve. The

absorbance of the solution was measured at the wavelength of 540 nm. In the standard curve, the

absorbance level increased at greater levels of protein concentration. Throughout the experiment,

the absorbance levels were used to determine unknown concentrations of protein on the standard

curve. In addition, an SpectroVis Plus was used to measure the absorbance levels of the colors in

the protein solution. This would be done after the biuret reagent was added to the protein

solutions in the test tubes. The SpectroVis Plus would then be used for each protein

concentration to examine the number of absorbance levels in the solution, which would

eventually measure the concentration of proteins in the solution. During the lab, Solution B,

Solution C, and Solution D were also diluted. This was to ensure that the amount of proteins in

these solutions would be less than the amount of proteins in the Isopure protein stock solution. If

undiluted, the amount of protein in the samples may exceed the highest standard point on the

standard curve graph. As a result, the data would not be usable for the lab experiment.

Lastly, the hypothesis of the study was that the protein would be detected in both the

solution with a dietary supplement protein powder, and in the solution of glycine amino acid.

The researchers predicted that both solutions would have a protein concentration in them and that

it would not be difficult to detect the protein in both solutions.

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Procedure

Materials

The experiments used in the lab included micropipettes, beakers, a waste beaker, test

tubes, a SpectroVis Plus, a LabQuest, two cuvettes, a Vortex, and laboratory tissue. Other

materials used included the Phosphate Buffered Saline, Biuret Reagent, and Bovine Serum

Albumin. In addition, a glycine solution, an Isopure protein supplement stock solution, and two

unknown solutions were used for the lab.

The micropipettes were used to measure the protein solutions. In this experiment, three

different types of micropipettes were provided; the P20, the P200, and the P1000. In addition, the

SpectroVis Plus and the LabQuest were used to measure the absorbance levels of the protein

concentration. Additionally, the Vortex was used to mix the protein solutions.

Methods

Prior to starting the experiment, the researchers tested the pipettes and practiced using

them to ensure that they were used correctly. After testing the pipettes, the researchers collected

stock solutions provided in the laboratory. The researchers collected 40 mL of phosphate-

buffered saline solution, 25 mL of Biuret reagent, and 15 mL of Bovine Serum Albumin into

labeled beakers. The labeled beakers of each solution were then brought to the lab table. Once

the beakers of the solutions were brought to the lab table, the researchers placed different

amounts of Bovine Serum Albumin solution and saline solution into fourteen separate test tubes.

Each of the test tubes was labeled starting from 1 to 14. For each test tube concentration, there

was a duplicate of the test tube with the same amount of protein concentration. Afterward, the

tubes were mixed using the Vortex.

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The researchers then collected small amounts of five different protein solutions in five

separate beakers. The researchers collected 8 mL of Solution A, which consisted of an Isopure

protein supplement stock solution. The researchers also collected 8 mL of Solution C, 8 mL of

Solution D, and 8 mL of the glycine solution. The researchers also collected 6.3 mL of

Phosphate Buffered Saline and 700 μL of the Isopure protein into a beaker, which would be

labeled Solution B. Then Solution B was carefully mixed. The Glycine solution would be labeled

Solution G. After collecting the protein solutions, the researchers placed the beakers onto the lab

table.

The researchers labeled fifteen additional test tubes from numbers 15 to 29. The

researchers pipette 2 mL of stock Isopure A into test tubes 15 to 17. The researchers then pipette

2 mL of solution B into test tubes 18 to 20. Afterward, the researchers pipette 2 mL of solution C

into test tubes 21 to 23. In addition, the researchers pipette 2 mL of Solution D into test tubes 24

to 26. Lastly, the researchers pipette 2 mL of Solution G into test tubes 27 to 29. Afterward, 0.7

mL of Buiret solution was added to all 29 test tubes. Then, each test tube was vortexed to ensure

that the protein solution and Biuret solutions were well mixed. The researchers then allowed the

protein to react with the Biuret solutions for 20 minutes at room temperature.

The researchers then turned on LabQuest and plugged in the SpectroVis Plus. Then they

changed the wavelength to 540 nm and calibrated the spectrometer. After the 20-minute mark,

the researchers placed test tube 1 into the cuvette. The cuvette was then placed into the

spectrophotometer. After the calibration was finished, the researchers made sure that the red box

said that there was a 0.0 absorbance value and removed the cuvette from the SprectroVis Plus.

The solution from the cuvette was then poured back into the test tube 1. The researchers then

poured the contents from test tube 2 into another cuvette and wiped the outside of the cuvette
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with a laboratory tissue. Afterward, the cuvette was placed into the SpectroVis Plus and the

absorbance values were recorded. The cuvette was then removed and discarded into the waste

beaker. The cuvette was then rinsed with water and the researchers reread the solution from test

tube 1. The researchers then poured the contents from test tube 3 into a cuvette. The cuvette was

wiped with laboratory tissue and then placed into the SpectroVis Plus. The researchers then

recorded the absorbance value, removed the cuvette from the SpectroVis Plus, and rinsed the

cuvette. This process was repeated for all the test tubes.

After recording the absorbance values for all the test tubes, the researchers plotted the

absorbance values to make the standard curve graph. The graph included a smooth-fit line or a

curve, to best fit the data. The researchers then determined the line equation for their standard

curve. Additionally, the researchers calculated the average absorbance of each protein sample

and the standard deviation between the replicates. The researchers then estimated the amount of

protein in each unknown sample by using the standard curve graph. In addition, the researchers

took note of the dilution factors in each sample and calculated the amount of protein in each

solution.

Results

Figure 1 presents a table of Absorbance Readings that is used for the standard curve

using the first 14 test tubes of the lab. After 20 minutes, the test tubes revealed that higher

concentrations of absorbance levels indicated that there were higher protein concentrations in the

solution. Figure 2 presents the absorbance values from Figure 1 plotted into the standard curve.

In addition, Figure 3 presents a table of the absorbance levels of the unknown protein

concentrations. Lastly, Figure 4 presents a table determining the protein concentration in

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Solution A, Solution B, Solution C, Solution D, and Solution G. The line equation used in the

standard curve was y = 0.1366x - 0.1895.

In addition, the researchers determined that there was 25.8 mg / mL of protein in the IP

stock solution. This was done by converting the 3 grams of Isopure powder in the 100 mL of the

phosphate-buffered saline Solution into milligrams, then multiplying the number by 0.86. This

was because 86 percent of the Isopure Powder contained proteins.

Figure 1

Absorbance Readings of the Standard Curve

Protein Concentration Absorbance Average Absorbance

0.00 0.00 0.004 0.002

0.4 0.078 0.070 0.074

0.8 0.185 0.145 0.165

1.6 0.300 0.306 0.303

2.4 0.542 0.522 0.532

3.2 0.660 0.652 0.656

4.0 0.825 0.774 0.7995

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Figure 2

Standard Curve Graph for the Average Absorbance Reading and Protein Concentration

Figure 3

Absorbance Readings for Unknown Protein Solutions

Samples Absorbance Absorbance Absorbance Average Standard

Absorbance Deviation

Isopure Stock 1.782 1.791 1.811 1.795 0.015

Solution A

Solution B 0.673 0.678 0.667 0.673 0.006

Solution C 0.248 0.255 0.262 0.255 0.007

Solution D 0.002 0.003 0.008 0.004 0.003

Solution G 0.001 0.000 0.013 0.005 0.007

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Figure 4

Table Determine the Protein Concentration in Isopure Solutions

Samples Protein Protein Dilution Amount of

Concentration Concentration Factor protein in
estimate by looking determined by using Solution A
at graph the line equation

Solution A 14.00 mg/mL 14.53 mg/mL XXXX 14.53 mg/mL

Isopure Stock

Solution B 6.0 mg/mL 6.31 mg/mL 10 60.31 mg/ mL

Solution C 3.2 mg/mL 3.25 mg/mL 20 65.00 mg/mL

Solution D 1.4 mg/mL 1.42 mg/mL 100 142.00 mg/mL

Solution G 1.41 mg/mL 1.42 mg/mL XXXX XXXX

Conclusion

The standard curve indicates that the higher the absorbance value, the higher the

concentration of protein would be found in the protein solution. In addition, despite using the

standard curve to calculate the protein concentration in the solution, the researchers did not make

accurate calculations for the protein concentration in each solution. This indicated that there was

some limitation to using the standard curve. As a result, the standard curve could only be used

for a small amount of data, rather than large amounts of data and for future predictions. In

addition, the standard curve could only be used for data with linear relationships, and could not

be used to calculate exponential data.

As for the glycine solution, no proteins were detected in the solution because it only

consisted of amino acids that did not form any bonds with one another. Therefore, there were no

proteins in the glycine solution. This can be seen in the lab when the biuret reagent does not turn

into a purple color in the presence of the glycine solution. This is because the biuret reagent only
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changes color when there are proteins detected in the solution. As for Solution A, Solution B,

Solution C, and Solution D, proteins were detected in the solutions. This is indicated through the

change of color in the biuret reagent in the solutions. Therefore, we can conclude that the

hypothesis was not fully supported by the results of the lab.

Unfortunately, the amount of protein in Solution A did not equal the expected amount of

proteins in Solution B, Solution C, and Solution D. As can be seen from Figure 4, the solutions

with the closest measurements to the expected amount are Solution B and C. In addition, much

of the data in the unknown protein solutions did not match the expected values. This may be

because of the influence of the dilution factors and it may not be accurate to use a standard curve

to determine the values for large varieties of data. In addition, this may have been caused by

errors in the data and the absorbance readings. This could have been a result of bubbles in the

micropipettes or inaccurate readings of the spectrophotometer. In addition, the disparities

between the expected values and the data could be a result of human error and miscalculations

done by the researchers.

Overall, this lab experiment is important for the researchers to understand how to find

and measure protein concentrations because proteins play an important role in the human body.

This may be crucial for researchers in understanding and studying concentrations of proteins in

food and nutrition, the amount of proteins needed to function in certain areas of the body, and

understanding the different functions of a protein. In addition, this may be crucial for researchers

and scientists who want to understand and learn more about the Biuret method and the standard

curve. Some suggestions for future research would be learning to fully utilize the standard curve

and the Biuret method using proper data and proper conditions to ensure that there are no

additional implications or errors that could occur during the experiment.