Running head: Measuring Protein 1

Measuring Protein in solution

Arizo J Niah

Stockton University

INTRODUCTION.

This experiment is to measure the amount of protein in several different given solutions. This

experiment is based off of the idea of how food manufacturers by law, must test to see how much

protein is in their products. To do so, research scientists conduct a similar experiment using

various protein assay methods. The protein assay method used in this lab was the Biuret protein

assay. Biuret is formed when urea is heated to 180 °C. This Biuret molecule has an amine group

in the center, and when four of these amine groups interact with one another, a purple color is

produced (which can be measured accurately and numerically with the SpectroVis). The purple

color is the result of copper ions binding (at a basic pH), which is made possible thanks to the

amine groups in Biuret. The more intense purple a solution becomes, the higher the

concentration of protein there is. The more opaque purple a solution is, the less concentration of

protein there is. Since this experiment calls for measuring protein in solutions where the amount
Measuring Protein in Solution 2

of protein is not known, using a solution with known protein is crucial. Using the data received

from the known protein, a standard curve can be produced to help with the computation of the

protein in other substances where the protein amount is unknown.

Materials and Methods

The lab started off with making sure everyone was well prepared before starting. This meant that

everyone had safety goggles and gloves on. After that was situated, six 50mL beakers were filled

with all of the indicated protein solutions. The first beaker had 40mL of Phosphate buffered

Saline (PBS). The second beaker had 19 mL of Biuret reagent (BR). The third had 15mL of

Bovine serum albumin solution (BSA). The fourth had 3mL of nonfat milk solution (NFM). The

fifth beaker had 3 mL of diluted egg white mixture (EW). The sixth and last beaker was filled

with 7 mL of glycine solution (G). Each solution was measured and put into the beakers using a

5mL pipette.

Next, 14 test tubes were obtained and filled with fixed amounts of BSA and PBS to create a

certain concentration using a table that was already provided. These were the protein assay

standard curve samples. First the PBS was pipetted (using micropipettes), and then the BSA

(known protein) was added afterwards. The micropipette tips were renewed after every change

in solution. The same tip was not used for two different solutions, to avoid contamination.

Vortexing the solution was also important afterwards as it assures that the solution is mixed. Test

tubes 1-14 were our known protein solutions and are what we used to base our standard curve on.
Measuring Protein in Solution 3

After that, dilution of the unknown protein samples was begun. Fixed amounts of each protein

was mixed and diluted with PBS into 12 more obtained test tubes, all labeled numbers 15-26.

Quickly afterwards, Biuret solution was added into all 26 total test tubes. When that was done,

they were set aside and left alone at room temperature for 20 minutes. While waiting for the

solution to incubate, the Spectrometer was set up and calibrated.

The spectrometer was used to collect absorbance data. Cuvettes were filled ¾ full with solutions

from each test tube, then measured for absorbance data using the spectrometer. Rinsing the

cuvette and shake drying it was important between each sample. All of the absorbance data was

collected and recorded into a table, which was organized by test tube number. Again, test tubes

1-14 were of the known protein samples, while test tubes 15-26 were of the unknown protein

samples.

Afterwards, data was analyzed by using the data in the tables. Using the data received from test

tubes 1-14, a standard curve was made (see below). The standard curve graph then helped aid in

getting the estimated values of protein in the unknown samples.

Measuring Protein in Solution 4

Results

DATA FROM TUBES 1-14 PLOTTED::

FINDING EQUATION LINE OF BEST FIT FOR STANDARD CURVE:

Measuring Protein in Solution 5

STANDARD CURVE EQUATION PLOTTED:

Discussion

In this experiment, we did have duplicates for our sample measurements. These

duplicates were in the same number range for the most part. There were a few small instances

where the numbers were oddly different. For our measurements of the second egg white sample,

one of the absorbances was .050 (The other 2 absorbances were .544 and .517). Obviously,

something was off there, which makes sense seeing that the standard deviation for that number

set was pretty large compared to the other sets.

The protein values we observed for the unknown sample were not that close to those of the

expected sample. An error was most likely made in the process. The numbers were not vastly

differently, but they were still a good amount off. One of the calculations could have been off,
Measuring Protein in Solution 6

throwing our whole data off as most of the data was cumulative when it came to processing

everything out. The estimations could have been inaccurate perhaps.

The Biuret reagent did turn a blue color in the presence of 1% solution glycine. Though glycine

is not a protein, it does have amino acids which are the building blocks of protein. People may

think that it reacts with the glycine because of this, but it does not, because the amino acid does

not have multiple peptide bonds. So in conclusion, the solution stays blue when mixed with the

Biuret reagent.

The first egg white dilution gave the best results of the expected value. This might be because the

solution was diluted the least out of the two egg white solutions.

If the standard curve was not linear, it most likely could not be used to accurately determine

other samples of protein concentration. Standard curves are linear because they are essentially

representing the average of a specific set of data points. When an average is represented, it

should have a slope that makes it linear and apply to all of the points.