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Protein Assay Lab Report

The Identification of Two Unknown Substances by Analyzing Protein Concentrations through the Usage of the Bradford Assay

By: Jane Wang

AP Biology

ABD Days, Periods 1-2

Mr. Resch

September 26th, 2008


            Biophotonics is the study of the interaction of light with biological material (Lague, 2005). Through the use of light, many properties of living and nonliving things can be discovered. Among those things possible is the identification of a substance by its protein concentration as determined from spectrophotometry and the usage of a Bradford assay. The purpose of this lab was just that – to identify two unknown substances by their calculating their protein concentrations and then comparing them to a table of known values.

            In the Bradford assay, we used the dye Coomassie G-250 which binds to proteins mostly at arginine but also at tryptophan, tyrosine, histidine and phenylalanine residues (Olson, 2007). When the dye is allowed to interact with a substance that contains protein, the arginine groups of the protein bind to Coomassie. This converts Coomassie G-250 which reaches its maximum absorbance potential at 595 nm and turns blue (Olson, 2007). Coomassie without the influence of protein is a brown color. Using these properties, we were able to prepare a set of standards using known protein concentrations. To calculate absorbances of the standards and the unknown samples, we used spectrophotometry. A spectrophotometer consists of a spectrometer to produce light of a specific color and a photometer to measure the intensity of the light (Caprette, 2005). Therefore, if a container (or more formally, a cuvette) of liquid of some opacity is placed between the spectrometer and the photometer, the photometer reader would change depending on the amount of light able to pass through the cuvette and the absorbance level could be recorded. Using these methods, we were able to measure the absorbances of the standards, plot them, and use them as our basis for concluding the identities of two unknown substances.





Table 1: Absorbances of solutions with various protein concentrations. Solutions were produced by mixing Coomassie G-250 dye with protein and were allowed to sit for five minutes before data was recorded.



Concentration (mg/mL)
































Figure 1: A graph of the protein concentration versus the absorbance of the resulting protein and dye solution. All seven standards, but no unknowns, are shown.


            After finding the absorbances of the standards (table 1), we plotted the concentrations of the standards versus their absorbances (figure 1). From this, we discovered a positive correlation between the concentration of the standard with its ab

sorbance. The R2-value of 0.8586 indicated a moderately strong correlation between the two variables. We used this data to calculate the protein concentrations of the two unknowns.

            By using computer software, we were also able to calculate the equation for a least squares regression line, otherwise known as a line of best fit. From that equation, we could estimate the protein concentration of each of the unknown samples from its absorbance:


            Now that we had the protein concentrations of the two unknowns, we could compare our data with the known data of five possible identities of the unknowns (shown below in table 2).


Table 2: The protein concentrations of five possible identities of the unknown substances.



Protein Conc. (g)

Serving Size (mL)

Concentration (mg/mL)

Silk Soy Milk




Red Bull




Whole Milk




Choc. Milk




Half and Half





However, we originally diluted our samples in a 1:50 ratio with PBS, so we should multiply our calculated concentrations by 50. That gives sample A a protein concentration of 81.5 mg/mL and sample B a protein concentration of 91.0 mg/mL. Since these concentrations are not near any of the concentrations on the list, we resort to other methods to determine what the unknown substances were. From qualitative observation, we noticed that sample A was a white color, while sample B was a tan color. This rules out chocolate milk for both of the samples. Also, the ratio of protein concentrations of A to B is 81.5/91.0 = 0.895. This ratio is closest to the ratio of the concentrations of Silk Soy Milk to Whole Milk/Half and Half (which is 29.2/33.3 = 0.877). We therefore conclude that sample A is Silk Soy Milk, while sample B is either whole milk or half and half.




            In this experiment, we wished to discover the identities of two unknown liquids by analyzing their protein concentrations through spectrophotometry. Since the dye we used changed to darker colors when in contact with a higher protein concentration, a higher absorbance meant a higher concentration. From this, we were able to estimate the protein concentrations of our two unknown samples and compare them to a list of known concentrations.

However, our calculated protein concentrations were far off from the given concentrations of possible substances. The amount of error found in our results could have been because of human error. Also, our R2 value was below 0.90, indicating that we did not have a very strong correlation between concentration and absorbance. This might have been because of impurities in our chemicals or lab equipment, improper procedures, smudges on glass, or other causes. Since our calculated concentrations were far off from the given concentrations by so much, we used qualitative analysis and analysis of the ratios of the concentrations to conclude that sample A was silk soy milk and sample B was either milk or half and half since the two had the same protein concentrations.





Caprette, David R. (May 19, 2005). Spectrophotometry. Retrieved September 25, 2008 from Rice University

                Web site:

Lague, Andrew. (November 08, 2005). Introduction & Basics. Retrieved September 25, 2008 from NSF Center

                for Biophotonics Web site:

Olson, Brad. (2007). The Bradford Assay. Retrieved September 25, 2008 from UNL Course Global Gateway

Web site:


This web page was produced as an assignment for an AP Biology course at Montgomery High School.
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Jane Wang,
Sep 25, 2008, 9:00 PM