Chem 436 Exp 4.1 Lab Report PDF

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Lab report for Experiment 4.1 Protein Assays
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Experiment 4.1 Protein Assays

Abstract Protein assays can be used to determine protein concentrations of unknown samples. The Biuret, BCA, Lowry, and the Bradford were used in this experiment. The goal of the different protein assays was to generate a standard curve of protein concentrations in order to determine the protein concentrations of

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unknown solutions. It was found that all protein concentrations were relatively the same for the unknown throughout each method. Introduction Protein assays can be used to determine protein concentrations of unknown samples. The Biuret, BCA, Lowry, and the Bradford methods are just a few protein assays. In the Biuret method, Cu2+  binds to the amide bonds in proteins in alkaline solutions. The coordination of Cu2+  to four nitrogen, two nitrogen from each polypeptide chain, causes the biuret solution to take on a purple color. Using this method, the protein Cu2+  complex absorbs at 550nm. An advantage of the biuret method is that there is little interference from free amino acids. However, a large quantity of protein is required for this method and, therefore, is the least effective method due to being less sensitive compared to other methods. The BCA method is a modification of the biuret method in which Cu+ , a by-product of the Cu2+  in alkaline solutions, is detected. Aromatic amino acids in proteins will reduce Cu2+  to Cu+ ions, which forms a complex with BCA (Bicinchoninic Acid), and this complex absorbs light at 560nm. The amount of Cu2+  reduced is proportional to the amount of protein found in solution. This assay is very sensitive and can detect very small quantities of protein. However, EDTA, DTT, and ammonium sulfate can interfere with BCA which puts this assay at a disadvantage. The Lowry method is also a modification of the Biuret method in which Cu+ reacts with phosphomolybdate-phosphotungstate. Aromatic residues such as tyrosine and tryptophan reduce Cu2+  to Cu+ which can then reduce the salt phosphomolybdate-phosphotungstate to an incredibly blue complex. This solution absorbs maximally at 750nm. The concentration of protein in solution is related to the concentration of tryptophan and tyrosine residues on the proteins. This assay is highly sensitive and optimal for reading low concentrations of protein. However, free aromatic amino acids can produce false positives and ammonium sulfate, glycine, zwitterions, and BME(beta mercaptoethanol) can interfere with this assay. The Bradford method employs the use of Coomassie brilliant blue dye which binds to protein causing a shift in dye absorbance from 465nm to 595nm. Under acidic conditions, the red form of this dye binds to the protein and changes its color to blue. The dye shifts the maximal absorbance wavelength from 465 (red) to 595 (blue). Therefore, the amount of protein in solution can be determined by the color change from red to blue. This assay is four times as sensitive as the Lowry assay since it does not rely on tyrosine or tryptophan and can determine protein in the range of 1-20 uL. Although, strong basic buffers and detergents can interfere with the Bradford assay. Using these four assays, standard curves can be generated of protein concentration and absorbance values. These curves can then be used to determine the protein concentrations of unknown solutions. Methods Before performing the assays, eight standards were prepared using the serial dilution technique. 10M BSA was diluted with distilled water to create standards. The standards ranged from 0.0078 to 1 mg/mL. Biuret Assay: Eight samples were created using .5 mL of BSA standards and 2.5 mL of biuret reagent while a ninth sample was created using .5 mL of the unknown protein solution and 2.5 mL of biuret reagent. Each solution was left at room temperature for 30 minutes. The absorbance of each was found at 550 nm, before each reading the spectrophotometer was blanked using 2.5 mL of distilled water. BCA Assay: Eight samples were created using .1 mL of BSA standards and 3 mL of BCA working reagent. A ninth sample was created using .1 mL of our unknown and 3 mL of BCA working reagent. Each sample was

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left to incubate at 37 °C for 30 minutes. The absorbance of each sample was read at 560 nm. Before each reading the spectrophotometer was blanked with .1 mL of distilled water and 3 mL of BCA. Lowry Assay: Eight samples were created using .5 mL of BSA standards, 2.5 mL of Lowry I, and 2.5 mL of Lowry II. A ninth sample was created using .5 mL of our unknown, 2.5 mL of Lowry I, and 2.5 mL of Lowry II. The absorbance of each sample was measured at 750 nm, blanking with Lowry I and Lowry II before each measurement. Bradford Assay: Eight samples were created using 3 mL of Bradford dye and .1 mL of BSA standards. A ninth sample was created using .1 mL of our unknown and 3 mL of Bradford dye. Each sample was left to incubate for ten minutes at room temperature. The absorbance of each sample was measured at 595 nm and the spectrophotometer was blanked with 3 mL of Bradford dye before each reading. Results/Discussion The purpose of performing the different protein assays was to develop a standard curve using known standard concentrations and the determined absorbance values. These curves were used to calculate the protein concentration of the unknown protein solution. For the biuret assay, the slope equation of the best fit line was found to be Y = 0.0475x + 0.0044 (See Fig 1). This equation was used to find the unknown concentrations by plugging in the unknown absorbances into the y value and solving for x. Unknown A had an absorbance of 0.057 and the protein concentration was calculated to be 1.12 mg/mL.

Figure 1. Graph showing the standard curve generated using the biuret method. Absorbances read at 550nm. The equation of the standard curve was found to be (Y = 0.0475x + 0.0044)

For the BCA assay, the equation of the best fit line was found to be Y = 1.4721x + 0.0436 (See Fig 2). Unknown A had an absorbance of 1.62 and a protein concentration of 1.07 mg/mL.

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Figure 2. Graph showing the standard curve generated using the BCA method. Absorbance read at 560nm. The equation of the standard curve was found to be (Y = 1.4721x + 0.0436)

For the Lowry assay, the equation of the best fit line was found to be Y = 1.3746x + 0.0471 (See Fig 3). Unknown A had an absorbance of 1.95 and a protein concentration of 1.39 mg/mL.

Figure 3. Graph showing the standard curve generated from the Lowry method. Absorbance values were read at 750nm. The equation of the standard curve was found to be (Y = 1.3746x + 0.0471)

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For the Bradford assay, the equation of the best fit line was found to be Y = 0.3985x + 0.0748 (See Fig 4). Unknown A had an absorbance of 0.448 and had a protein concentration value of .937 g/mL.

Figure 4: This graph shows the standard curve for the Bradford method. The absorbances were read at 595nm. The equation of the standard curve was found to be (Y = 0.3985x + 0.0748)

All 4 of the assays show a general upward linear trend on all standard curves. This means that as concentration increases, absorbance increases. The Biuret assay is the least sensitive method for protein determination which is supported by the data because the biuret assay yielded the lowest absorbance values for the unknown. The BCA assay and the Lowry assay are more sensitive than the Biuret assay. The absorbance values found for these two methods should be higher than the absorbance for the Biuret assay which is supported by the data. The calculated protein concentrations for the unknown are relatively the same throughout each method.

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References 1. Ninfa, Alexander J., and David P. Ballou. Fundamental Laboratory Approaches for Biochemistry and Biotechnology. John Wiley & Sons, 2015....