Bradford Assay: Experiment 5 Lab Report Guide

BIOL 3380 Experiment 5 - Lab Report Bradford Assay Name: Lab section: Instructor: Jing Pan Graduate TA: Date: 10/23/2023 5-1

Experiment 5 – Lab Report Bradford Assay Edit this word document by inserting your answers after each question/space. Please denote your answers by using bold , italic s, or colored font. Do NOT renumber/reorganize the questions. “Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of your handwritten work, or using handwritten annotation on the document (if you have the technology to do so). Insert graphs (handwritten on graph paper or electronically constructed with horizontal/vertical gridlines) as a clear image with appropriate titles, labeled/scaled axes, and standard deviation bars when appropriate. 1. (4pts) Attach the image of the BSA standard curve you constructed in the lab. All figures/graphs/tables must include appropriate titles, legends, labeled/ scaled axes, and 5-2

standard deviation bars (when applicable). 5-3

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2. (1pt) Attach images of all microplate data sheets. 5-4

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3. Determine the protein amounts and concentrations based on your results from the Bradford assay. a) (3pts) What is the total amount of protein in each W4 trial ? If using a hand-drawn standard curve, mark the three interpolation lines and include the image in your answer. Alternatively, you can construct a standard curve using apps such as Excel or Google sheet. If you use a computer-generated standard curve, include both the standard curve graph (label axes properly) and the trendline equation in your answer. 7.6 ug, 9.4 ug, 8.6 ug 5-6

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b) (2pts) What is the total amount of protein in the 500ul W4 fraction ? Calculate the average and standard deviation. Show your work. d) (1pt) What is the protein concentration of the W4 fraction? Calculate the average and standard deviation. Show your work. 4. (1pt) Make a table of the total amount of protein present in 500 ul column fractions W1-W6 and E1-E6 calculated from the Bradford assay. 5. (5pt) Based on your table from Q4 above, construct a “combined elution/activity profile” that depicts both the rGFP activity (determined in the previous Experiment 4) and the total 5-8

amount of protein present in each fraction. Examples of bar graphs were shown in the lecture note. Include appropriate titles, legends, labeled/scaled axes, and standard deviation bars (when applicable). 6. The ratio of an enzyme activity and the total amount of protein in that enzyme preparation is known as the “specific activity”. This ratio intrinsically takes in account the differences in purity and activity between different enzyme preparations/purifications. For rGFP, we will define the specific activity as the relative fluorescent units per mg of total protein. a) (2pts) What is the specific activity of your E4 fraction? Show your work for credit. E4 = b) (2pts) If the TA also performed the same experiment and found a lower specific activity in their E4 fraction, who (you or the TA) had the purer fraction? I had the purer fraction if the TA conducted the same experiment and discovered a lower specific activity in their E4 fraction. This is due to the fact that specific activity is a gauge of purity and immediately relates to purity. Purity rises in tandem with an increase in a particular activity. 7. (3pts) What would happen to the purity, yield, and activity of rGFP if you pooled (i.e., combined) the E2 and E3 fractions and then applied the pooled fractions onto a new Ni +2 - agarose column? Because we would be "following a seal act with a seal act," or continuing to purify using the same property—the affinity of the GEP's His tag to the Ni+2—as we had done in the previous Ni+2-agarose column, the purity would remain unchanged if I combined the E2 and E3 fractions and then applied the pooled fractions onto a new Ni*2-agarose column. Because the Nit?-agarose column is not 100% efficient, the yield will decrease. As a result, lost yield and lost proteins would probably occur. Because a lower yield would result in less activity, there would be less activity. 8. The food industry commonly determines protein concentration using an acid hydrolysis assay that measures nitrogen content. In illegal attempts to increase the apparent levels of protein milk products have been adulterated with melamine and cyanuric, which are inexpensive and rich in nitrogen. Melamine and cyanuric acid poisoning in infant formula led to the hospitalization of over 50,000 children with kidney stones in China in 2008. At least 6 deaths occurred. To find an alternative protein assay suitable for food products, the Bradford experiments below were performed (adapted from Field and Field, Food Chemistry. 2010. 121(3):912- 917) 5-9

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(1pt) Looking at Figure A above, purified BSA (bovine serum albumin) and reconstituted- powdered milk at various protein concentrations were measured using Bradford assay. A significant difference is observed between the BSA and the milk curves. For example, at 10 µg/ml, BSA had an A 595 of ~0.6, whereas milk had an A 595 of only ~0.3. Provide an explanation on why milk and BSA have very different A 595 readings when tested at the same concentration. Looking at Figure A, the discrepancy between the BSA and the milk curves may be due to the fact that the reconstituted powdered milk contains cyanuric acid and melamine, while the BSA has more pure proteins. The Bradford Assay would not account for this and produce an increase in absorbance readings since it would not read the cyanuric acid and melamine. Despite melamine's abundance in amino groups, the Coomassie blue dye may not necessarily bind to it because it binds to primary amines and carboxyl groups and lacks a hydrophobic side chain in addition to having an aromatic ring. Furthermore, distinct protein quantities, as well as proteins found in milk and bsA, might have various molar extinction coefficients at a given wavelength. Different absorbance levels may result from differences in molar extinction coefficients and protein concentrations, even though the A595 readings are evaluated at the same concentration. A B C D E F G 5-10

Looking at Figure B, various combinations of milk, melamine, and cyanuric acid were tested using the Bradford assay. (1pt) Comparing assays E through G, what can you conclude about the detection of melamine by the Bradford assay? I can conclude from comparing assays E through G that the Bradford assay is inaccurate for detecting melamine since it is unable to do so. When we look at assay E, we can see that the only protein introduced was melamine, and the absorbance value was 0. (1pt) Comparing assays E through G, what can you conclude about the detection of cyanuric acid by the Bradford assay? By contrasting assays E through G, I am able to determine that the Bradford assay is inaccurate in its detection of cyanuric acid due to its inability to detect the substance. As we can see from experiment F, the only protein provided was cyanuric acid, and the absorbance value was zero. (1pt) Using the Bradford assay, cyanuric acid does not significantly interfere with the detection of protein in milk samples. Comparison of which two assays supports this conclusion? The finding that cyanuric acid does not appreciably obstruct the Bradford Assay's ability to identify protein in milk samples is supported by a comparison of assays A and C. Both Assay A and Assay C have the same absorbance values and contain milk and cyanuric acid, respectively. (2pts) Considering the standard acid hydrolysis assay used by the food industry, explain why the Bradford assay is the assay of choice when testing milk samples potentially adulterated with melamine and/or cyanuric acid. When assessing milk samples that may have been tampered with using cyanuric acid or melamine, the Bradford assay is the preferred method because the addition of these substances has no effect on the absorbance value. As a result, the Bradford assay is perfect for determining the actual protein concentration in milk and demonstrating that adding cyanuric acid or melamine does not raise the protein content. However, because the usual acid hydrolysis assay only evaluates nitrogen concentration, which is present in both proteins and melamine/cyanuric acid, it is unable to distinguish between the pure genuine proteins from milk and these substances. 5-11

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Exp 5 Lab Report F23

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