6-PAGE_report_2023

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Colorado State University, Fort Collins *

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Jan 9, 2024

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LIFE 212 Laboratory Report 6 PAGE Name: _____________________________ Due: ___________________________ Section: _____________________________ 100 pts total plus Notebook Conclusion (4 pts) Characterization of Proteins Part 1: Protein Separation by Gel Electrophoresis (60 points) 1. (10 pts) In the space below, paste a picture of your gel that was taken by the gel documentation system. Label all the gel lanes, and sizes of each MW standard band. Label the putative tyrosinase band on this gel. Circle a single band of your choice. 2. (10 pts) Calculate the R f ’s for the bands of the protein standards and for tyrosinase (for the R f of tyrosinase, use the most purified lane, PPT). Use this data to complete the table below. 101 Protein MW (Daltons) Log 10 MW R f 1 250,000 5.397 2 3 4 5 6 7 8 9 10 10,000 4 Tyrosinase

3. (10 pts) Plot and paste in the space below an Excel graph of log 10 MW versus R f for the bands of the protein standards. Include your data sheet with the graph chart. See Kendall-Hunt Appendix II for instructions on plotting logarithmic MW data and Appendix I for plotting linear graphs. 4. (5 pts) Using the formula that you generated from the standard curve in the graph above, determine the MW of tyrosinase. Examine the bands between the potato homogenate lane and the partially purified tyrosinase lane to find one major band that is enriched in purity compared to the other bands within the lane, choose this band. Whichever band you choose is okay, as long as it is marked on the gel image. Show your work for determination of tyrosinase molecular weight and R f value. 102

LIFE 212 Laboratory Report 6 PAGE Name: _____________________________ Due: ___________________________ Section: _____________________________ 5. (6 pts) Refer back to the procedure used to prepare tyrosinase enzyme from the Enzymes I lab. We prepared a potato slurry in sodium fluoride/ammonium sulfate to make the potato homogenate. Then we pelleted the precipitated protein to enrich for tyrosinase. We expected that the dual spin steps increased purity of tyrosinase. Did this occur? Why or why not? Explain what we could have done to improve the quality of the enzyme purification. 6. (13 pts) Explain the purpose of the ingredients of the polyacrylamide gel (5 pts). Which ingredients affect the speed of the polymerization of the gel (4 pts) , and which ones affect the resolution of the protein bands (4 pts)? 7. (6 pts) What could cause a protein to migrate on SDS-PAGE in such a way that it appeared to be a protein that was much bigger than its actual size? List and explain three technical reasons related to the treatment of your protein sample before loading on the gel (2 points each). 103

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Part 2: Determining Protein Concentration: Bradford Assay (40 points) 1. (10 pts) Protein Concentration Data: Sample Concentration μg/mL Amount in μg A 595 Standard #1 Standard #2 Standard #3 Standard #4 Standard #5 Standard #6 Unknown ( ) Crude Potato Homogenate (H) Partially Purified Tyrosinase (PPT) 2. (10 pts) Plot and paste in the space below the standard curve from the data above relating A 595 versus BSA in micrograms. Label the axis, the graph title. Include the data sheet and the graph with the trendline and line equation. 104

LIFE 212 Laboratory Report 6 PAGE Name: _____________________________ Due: ___________________________ Section: _____________________________ 3. (8 pts) Using the formula that you generated in the standard curve above, what is the protein concentration (mg/ml) of your unknown protein sample? (4 points) What is the concentration of your crude homogenate and partially purified tyrosine? (4 points) Be sure to take any dilutions you made into account. SHOW YOUR WORK. My unknown protein sample is ______ (A, B, C, D, E, F, G). 4. (5 pts; Critical thinking question) List 5 ways that erroneous protein concentrations can arise when using the Bradford assay. 105

5. (7 pts) Starting with a 1 mg/ml solution of BSA, how would you prepare 100 microliters of each of the BSA standards (See question 1)? Show your calculations. Include appropriate units. 106

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LIFE 212 Laboratory Report 6 PAGE Name: _____________________________ Due: ___________________________ Section: _____________________________ Notebook Conclusion (4pts) The conclusion should be a concisely written summary of the experimental results using technical language. Describe each objective from your Introduction individually and frame in context with the experimental results. Vague and/or incomplete conclusions will not receive any credit. 107

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