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Lab Report 1: Separation of Proteins
Abstract/Summary: “Proteins account for more than 50% of the dry weight of most cells, and they are instrumental in almost everything organisms do” (Campbell, 1999). The significance of proteins to the continuation of our biological systems is undeniable, and a study of how to quantify proteins seems an appropriate introduction to our studies of biology. In order to study proteins we must first know how to separate then quantify the amount using basic principles of experimental design such as a standard curve. In this experiment we wish to quantify the amount of previously extracted protein by measuring the absorbance of the unknown amount and determining its concentration by overlaying it against a standard curve of the absorbance of known concentrations of the protein. We used the dye agent Bradford Protein Assay to get an absorbance of 0.078, 0.143, 0.393, 0.473, and 0.527 at the protein’s respective concentrations of 0.28, 0.56, 0.84, 1.12, and 1.40 mg/mL. When a best-fit line was applied to the standard curve, and the absorbance of our unknown concentration (0.317 A) plotted, we estimated a concentration of around 0.84 mg/mL of protein. Our calculations indicated a quantity of 168 mg of protein, which was an approximately 8.96% yield of the projected 1875 mg that was expected. Errors that may have led to this small yield percentage may have stemmed from our previous lab and our initial attempts to extract the desired amount of protein.
Introduction: Within this experiment we wish to facilitate a greater understanding of the concepts of experimental design and quantifying techniques. Specifically, this lab will allow us to gain an enhanced understanding of the isolation of a protein using differential solubility, which allows us to separate and purify various proteins using high concentrations of a specific salt so that they may be studied in great detail. Last week we separated our desired protein using ammonium sulfate. Since we have already extracted the desired protein, we will begin quantifying the amount using the Bradford Protein Assay. Because it is a dye-binding assay, we will use the spectrophotometer to measure the absorbance of various dilutions of a protein: this will comprise our standard curve. We will then compare the absorbance of our extracted protein from last week against our standard curve to allow us to first obtain the protein concentration, and then to quantify the amount of the protein.
Materials/Methods: We shall first prepare dilutions of the protein standard containing 0.28 to 1.40 mg/mL of Protein in test tubes labeled 1A-6A. We shall then transfer 0.1 mL from each of the dilutions in tubes 1A-6A into their respective tubes labeled 1B-6B. We then add 5.0 mL of Diluted Dye Reagent into these tubes (1B-6B) and mix gently. Afterwards we will transfer the contents of tube 1B into a cuvette as our blank and zero the absorbance. Similarly transfer the solutions from tubes 2B-6B into cuvettes and measure the absorbance to determine our standard curve at wavelength 595 nm (A595). After recording all your results into the first table and calculating the projected concentrations, plot the absorbance vs. mg of protein in your milk samples. For the second part of the experiment, first obtain the milk protein previously extracted from last week’s experiment. Since any ions dissolved into the solution could affect our projected results, we will first add a few drops of 2% barium chloride (BaCl2) to the test tube. If the sulfate ion did not fully diffuse through the dialysis bag, then “a white precipitate of barium sulfate (BaSO4) will appear although there should be none” (Lab Manual, 2001). Measure the volume of the dialysis tubing (it should already be pre-measured from last week, but the volume may have changed), and record. Place 0.1 mL of your protein sample into a labeled test tube. Zero the spectrophotometer with the contents from tube 1B (the blank), and measure the absorbance of your sample afterwards at wavelength 595 (A595). Using the standard curve that you previously constructed, plot the absorbance of your sample protein and find the approximate concentration of the protein sample. If the absorbance is way off your standard curve you will have to prepare dilutions of the protein sample, very similarly to the ones we prepared for the standard curve. Use the nutritional information from the back of the milk carton from which you derived the protein sample, calculate the amount of protein expected per mL of milk. After calculating the amount of protein extracted from your undiluted solution, now calculate the percent yield. Once you are done with the laboratory, be sure to dispose of all the waste properly. Rinse all the tubes with distilled water, remove any labels from those tubes, and place mouth down across the test tube racks. Clean all cuvettes with methanol and allow to air dry.
Results:
Standard Curve Preparation
Table 1
Tube Number Amount of Standard (mL) Amount of Water (mL) Protein (mg/mL) Concentration Absorbance Units (A)
1B 0.0 1.0 0.00 0 (blank)
2B 0.2 0.8 0.28 0.078
3B 0.4 0.6 0.56 0.143
4B 0.6 0.4 0.84 0.393
5B 0.8 0.2 1.12 0.473
6B 1.0 0.0 1.40 0.527
We used a best-fit line to set a standard curve for the absorbance of known concentrations of the protein. From there we plotted the absorbance of our sample protein (0.317 A) along that best fit line in order to find the corresponding protein concentration so that we could determine the amount of extracted protein in the calculations below.
Calculations:
Amount of Protein extracted (expected):
=(Concentration from curve)(dilution)(volume in tube)
=(9000 mg/mL)(1)(50mL)= 1875 mg protein
Amount of Protein extracted (actual):
=(Concentration from curve)(dilution)(volume in tube)
=(0.84 mg/mL)(20)(10mL)= 168 mg protein
%yield=[extracted/expected]100%
%yield=(168mg/1875mg)=(0.0869)100%= 8.96%
Conclusion: Aforementioned, proteins are an essential component in the processes that keep organisms alive. “Proteins are the most structurally sophisticated molecules known” (Campbell, 1999) which is reason enough to study them. The techniques we learned in this lab form a basis from which a detailed study of proteins is possible. Following our procedure we were successfully able to set up a quantifying assay to determine the amount of protein within a milk sample, although our yield percentage was rather low. However, errors in this lab (in the form of a low yield percentage) may have an origin from our last lab. In the process of extracting proteins from the milk sample, we may have inadvertently lost some of the protein through erroneous measurements, or perhaps through poor handling of either ammonium sulfate or the dialysis tubing. While not sufficient enough (at this point) to invalidate our results, they do explain the major difference between the expected and the actual amount of protein extracted.
References:
Laboratory Manual: Biological Sciences 112, University of California Department of Biological
Sciences, Fall 2001.
Campbell, N. A., Reece, J. B., & Mitchell, L. G. Biology: Fifth Edition. Addison Wesley
Longman, Inc. Menlo Park, 1999.
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