Calculating Protein Concentration Using Protein Standard Curve

Quantitative Test for Proteins2Introduction Proteins, a very important macromolecule, have many different function essential in the body. One of the most important role of proteins in the body is to help with muscle building and muscle recovery, which is the reason why protein powders are commonly used in fitness. It is important to know the concentration of protein within an unknown sample to determine how much protein is absorbed in the body and be able to design an effective workout plan along with the proper diet plan in order to achieve desired results in fitness.

The manufacturer of a protein powder called “Tough Guy” claims that if a person was to dilute 1 gram of protein powder in 100 ml of water, the final concentration of protein should be approximately 0.40 mg/ml (+/- 5%) protein. A procedure requiring the use of Bio-Rad protein assay will be conducted in order to create a protein standard curve to verify whether the manufacturer’s statement is correct. If the manufacturer’s advertisement is true, and a protein standard curve is used to calculate the concentration of the protein, then the final concentration of the protein should be approximately 0.40 mg/ml (+/- 5%).

Materials and Methods
One gram of Tough guy protein powder was added to 100 ml of water to serve as the unknown sample. 50 microliter (ul) of the unknown sample is added to the appropriate cuvette containing different concentrations of protein: 0.2mg/ml, 0.3 mg/ml, 0.6 mg/ml, 0.9 mg/ml. A cuvette containing just the sample was also prepared and a cuvette containing only water served as the negative control as well as to use to blank the spectrophotometer. 2.5 ml of diluted Bio-Rad assay was added to each cuvette. A small piece of paradigm was placed over the cuvette and vigorously shook for a few seconds. The samples sat in room temperature for 5 minutes, before measuring the optical density of each cuvette using a spectrophotometer set at 595 nm. The relationship between protein concentration and the absorbance was recorded on using an Excel spreadsheet.

Quantitative Test for Proteins3Results The experiment using 0.2, 0.3, 0.6, and 0.9 mg/ml of proteins yielded optical densities of 0.193, 0.252, 0.452, and 0.841 shown in Table 2. Figure 1 shows the protein standard curve of the protein concentration and their respective optical densities. From the graph, a positive linear trend-line shows that as the concentration of protein increased, the absorbance also increased.

Avg Absorbance (OD)00.2250.450.6750.9Protein (mg/ml)00.30.60.9y = 0.911x – 0.021R² = 0.9681Avg Absorbance (OD)Protein Standard CurveTable 1: Absorbance recording of each
protein (mg/ml) taken at 595 nm.Protein (mg/ml)Absorbance (OD)0.20.1920.20.1940.30.2520.30.2510.60.4460.60.4570.90.8480.90.833Unknown TG0.598Unknown TG0.596Protein (mg/ml)Avg Absorbance (OD)0.20.1930.30.2520.60.4520.90.841Table 2: Average absorbance of each protein (mg/ml)
taken at 595 nm. Does not include unknown
solution with Tough Guy.

Quantitative Test for Proteins4Discussion Using the protein standard curve, our calculated value for the concentration of the Tough Guy protein was 0.678 which was higher than the manufacturer’s value of 0.4 mg/ml. Different variables could have affected our results including not wiping off the cuvettes completely which gave inaccurate reading of the optical densities and human errors. Halfway through the experiment when adding the 2.5 ml of bio-rad assay solution, we ran out of solution and had to borrow another group’s solution. This delay in the experiment could have given us a different reading on the other cuvettes since they sat a bit longer than the others.

The lab manual also called for using a vortex, but we opted for that and just shook the cuvettes by hand, which also could have played a role in achieving the desired goal. For this reason, I would have to say that our experiment was inconclusive and another experiment should be performed avoiding the variables mentioned to see if data readings would change and if a more precise trend line and better correlation is possible.