Protein Synthesis Lab

Laboratory Exercise #3
Measuring Protein in Solution
Abstract
The purpose of this lab was to learn about the Biuret assay reaction to determine if it can detect proteins and amino acids; also, to understand the process of “salting out” proteins and how to determine the amount of protein in a solution. In order to do so, egg white and ammonium sulfate were mixed on ice and then put into the centrifuge. After PBS was added, the amount of protein could then be determined. After that, 14 test tubes were used to create a set of protein standards. Biuret solution was added to all 22 tubes and vortexed. Absorbance data was then collected by these protein standards using the SpectroVis Plus, and there was a direct relationship between absorbance
and concentration as shown in the graph.
Introduction
The purpose or objectives of this lab were to understand how to determine the amount of protein in a solution and how to “salt them out.” In addition, one objective was to see if the Biuret assay could detect proteins and amino acids in solutions. This lab is useful because research scientists often need to figure out the amount of protein in a solution, in order to do things like give nutritional facts about protein on food items, as required by law. While there are many methods to use, one of the simplest methods is the Biuret protein assay. This lab introduces the study of proteins which are macromolecules. Macromolecules are formed from smaller molecules made up of monomers. Amino acids are monomers that form proteins. Polymers are formed when the carboxyl group and amino group of two amino acids form a peptide bond. Water and dipeptide are formed in the reaction. Adding salt to a protein solution causes proteins to precipitate because salt has a greater affinity to the water molecule and pulls it away from the protein. Research scientists use the Biuret method to determine the amount of protein in a solution. In a certain recent study, the variation in protein color was measured at 540nm using the Biuret assay with bovine serum albumin as the reference protein. The colors varied greatly among the different gelatins because of the difference in amino acid content. Gelatins were then classified into 2 groups- 1 with all cold water fish gelatins and 1 with warm water fish, avian, and mammalian species (P. Zhou and J.M. Regenstein). In lab #3, a Biuret reagent was also used to determine the protein content of different solutions. Then, the measure of absorbance was used to create a standard curve which showed a direct relationship. Because the relationship is linear, protein concentration could be calculated using the line equation for the standard curve. Additionally in this lab, eggs were used with a variety of solutions to detect and measure protein.
Materials and Methods
The experiment was done exactly has written in the Cells and Molecules Laboratory Manual (20XX) on pages 37-42.
However, there was a typo in the lab on page 39, step number 10. It says to add 0.666uL of Biuret solution to all 22 test tubes, but it was supposed to say 0.666mL. Therefore, the correct results were provided to the class because the data the class recorded was done will 0.666uL of Biuret solution.
Results
Table 3
Protein Concentration (mg/mL) Absorbance Average
Absorbance
0.00 0 0 0
0.4 0.108 0.122 0.115
0.8 0.167 0.171 0.169
1.6 0.302 0.309 0.3055
2.4 0.550 0.542 0.546
3.2 0.707 0.708 0.7075
4.0 0.830 0.848 0.839
Due to the typo in step number 10 of the lab manual, the class received the wrong data values. The table above was given to us by the instructor in order to make an accurate standard curve that represents the correct results of this experiment. The graph was made with protein concentration in the X-axis and the average absorbance in the Y-axis, and a linear line was formed from the data, showing a direct relationship between absorbance and concentration. This graph was then used to determine the protein concentration from the line equation in Table 4.
Table 4
Samples Absorbance Absorbance Average Absorbance Protein Conc. From line eq. (mg/mL) Actual Conc. Adjusted for dilution factor (mg/mL) Concentration calculated from label or website (mg/mL)
1% Nonfat milk 0.344 0.342 0.343 1.57 15.7 42.28
EW1 0.959 0.962 0.9605 4.5 45 36
EW2 0.501 0.492 0.4965 2.3 23 36
Glycine/Tryptophan 0.06 0.045 0.0525 0.197 Discussion
The purpose of this lab was to determine if a Biuret assay could be used to measure the concentration of proteins and amino acids in solutions.
As clearly shown by the tables and the graph, the Biuret was useful in doing so- not only for known solutions, but also unknown solutions. In order to determine the concentration of protein in an unknown solution, the graph was used to see where the absorbance fell on the standard curve. In this lab, there was a typo in the lab, resulting in inaccurate results by the entire class. Therefore, the proper typical results of this experiment were provided by the instructor in order to get a better representation of the data in the experiment to be used for the standard curve. The Biuret assay is a very useful tool by scientists, particularly because it is used to determine the amount of protein in foods and drinks on the market, which is necessary because it is required by law to state the nutrition facts on the labels of all food products. The Biuret assay was also useful in the experiment done by P. Zhou and J.M. Regenstein, who used it to determine the concentration of protein in cold water fish gelatins as well as warm water fish, avian, and mammalian
species.
References
Wiley Online Library http://onlinelibrary.wiley.com/doi/10.1111/j.1750-3841.2006.00151.x/abstract;jsessionid=D399B37ADC528B4BAE145546BF0CCF5F.f04t04?deniedAccessCustomisedMessage=&userIsAuthenticated=false