BIOS 312 Homework 4 Sp 2023 (1)

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BIOS 312 Cell Biology Laboratory Homework 4 20 points Page 1 of 4 Name: ________________________________________________________________________ TA: ____________________ FALL SPRING SUMMER YEAR: ___________ Complete the following assignment related to the SDS-PAGE exercise. Homework must be submitted using the link provided in the Blackboard weekly folder before 11:59 pm on Friday of the week it is due. Upload your completed assignment as a PDF, DOC, or DOCX file. Do NOT submit a JPG image of your homework! This will not be accepted, and you will not receive any credit . Your assignment will be checked with the plagiarism detection software SafeAssign to verify that you did not copy the work of another current or past student in the course. Feel free to attach extra pages if you need more space. To complete this assignment, you’ll need the distance measurements and Rf values from the “Data analysis” portion of the SDS-PAGE exercise (page 4 in the lab handout.) You should have completed this during Week 4. If you didn’t, there is a link to the Control Gel image in the Week 4 folder. You will also need a metric ruler or a copy of the printable ruler, also available in the Week 4 folder. Follow the instructions in the SDS-PAGE handout. 1. Use Microsoft Excel or an equivalent spreadsheet application like Google Sheets to prepare a scatter plot of log 10 of molecular weight in daltons (Da) versus protein mobility (Rf) for each protein standard. Remember that on the Control Gel, molecular weights of the standards were expressed in kilodaltons (kDa), not daltons, so you’ll need to make the appropriate conversion first. You also need to calculate the log 10 of each molecular weight or have the spreadsheet do this. Format your graph according to the following guidelines: • Protein mobility (Rf,) the value you determined from your distance measurements, should be plotted on the Y axis. Rf is a ratio and does not have units. • Log 10 of molecular weight should be plotted on the X axis and also has no units. • A linear regression trendline should be plotted through your data points. Display the R 2 value (correlation coefficient) and the equation for the line on your chart. • An appropriate figure legend should be displayed BELOW your graph, not on the chart itself. The legend can be added after the graph is created and should include a title and a description of what the figure is showing. Use only data for the molecular weight standards, NOT the unknown protein bands, when creating your graph. If you’ve forgotten how to use Excel to make a scatter plot, consult the links at the end of this assignment. Hint: You can use the spreadsheet you created for absorbance versus concentration in Homework assignment 2 as a template. This time, the independent variable (the one you controlled) will be log 10 of standard molecular weight instead of standard concentration, and the dependent variable will be protein mobility (Rf) instead of absorbance. A sample graph is shown on the next page. Do not copy from it or you will lose points!

BIOS 312 Cell Biology Laboratory Homework 4 20 points Page 2 of 4 Figure 1. Protein mobility versus mass . Relative band migration distances for known protein standards on an SDS-PAGE gel were compared with protein mass. A linear relationship was observed between Rf and the log 10 of molecular weight (MW) in daltons. 2. Calculate the molecular weight (MW) of each unknown band from the Rf value you determined in the SDS-PAGE exercise. Using the equation for the trendline of the standard curve you created in step 1, enter the Rf value for each unknown as Y and then solve for X. Here, X will be the log 10 of molecular weight, not molecular weight itself, so you will have to make the conversion. If X = log 10 [MW], then MW = 10 X . 3. Fill in the missing entries in this table. You’ll need the distance and Rf values you determined in the “Data analysis” portion of the SDS-PAGE exercise and the molecular weights you calculated in step 2. Remember to convert molecular weights from Da to kDa. Table 1. Distance travled (mm)/ Molecular weight Band Lane Distance migrated (mm) Rf Molecular weight (kDa) Log 10[MW} 170 kDa standard 1 4 0.027 170 5.230448921 130 kDa standard 1 13 0.089 130 5.113943352 95 kDa standard 1 28 0.192 95 4.977723605 72 kDa standard 1 42 0.289 72 4.857332496 55 kDa standard 1 55 0.378 55 4.740362689 43 kDa standard 1 75 0.514 43 4.633468456 34 kDa standard 1 93 0.637 34 4.531478917 26 kDa standard 1 111 0.76 26 4.414973348 17 kDa standard 1 137 1 17 4.230448921 Unknown Band * 3 10.5 0.072 112 5.049218023 Unknown Band x 3 58 0.4 54 4.73239376 Unknown Band > 3 68 0.469 46 4.662757832 Description: The distance migrated is the total amount that the protein traveled. This is to be calculated by viewing the scale and SDS-PAGE protein. The Rf Value is the distance traveled by the protein/the distance traveled by the dye. The molecular weight is MW=10^x

BIOS 312 Cell Biology Laboratory Homework 4 20 points Page 3 of 4 Submit for grading 1. (10 points) Your XY scatterplot of log 10 molecular weight versus protein mobility (Rf) for the molecular weight standards. The two variables must be plotted on the correct axes [2 pts], and both axes must be appropriately labeled [2 pts.] A linear trendline should be plotted on the chart by the spreadsheet and should not be hand-drawn. The trendline must be based only on your log 10 MW and Rf data [2 pts.] A figure legend must be displayed below your graph [2 pts.] This legend should include a title [1 pt] and a description [1 pt] that explain what the plot represents. Do NOT simply repeat the title and description from the sample graph! You will receive no credit if you do this! 2. (10 points) Your completed Table 1 from the previous page. All missing entries must be filled in [2 pts.] The Rf values in column 4 must be reasonable given the positions of the protein bands and dye front in the Control Gel image [2 pts.] The calculated molecular weights of Unknown Bands *, x, and > must be reasonable based on the positions of the bands in the Control Gel image and should be expressed in the proper units [2 pts.] A title and description should be added to the table in the spaces provided [2 pts.] The title should indicate what data are presented in the table [1 pt], and the description should explain, in 1- 2 sentences, where those data came from – what experiment was done? [1 pt] Instructions for preparing XY scatter plot graphs in Excel: https://support.office.com/en-us/article/present-your-data-in-a-scatter-chart-or-a-line-chart- 4570a80f-599a-4d6b-a155-104a9018b86e Instructions for adding a linear trendline in Excel: https://support.office.com/en-us/article/add-a-trend-or-moving-average-line-to-a-chart-fa59f86c- 5852-4b68-a6d4-901a745842ad

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BIOS 312 Cell Biology Laboratory Homework 4 20 points Page 4 of 4 Calculations Rf= Distance traveled protein X= log 10^[mw] Distance traveled by front mw= 10^x Rf value for unknown band (*) 10.5 mm/150mm= 0.072 Unknown band (*) Y= -0.9679(0.072) +1.1203 = 2.05 10^2.05= 112.202 kDA Rf value for unknown band (x) 58mm/150 mm = 0.4 Y= -0.9679(0.4) +1.1203 = 1.733 10^1.733= 54.076 kDA Rf value for unknown band (>) 68 mm /150 mm = 0.469 Y= -0.9679(0.469) +1.1203 = 1.666 10^1.666= 46.345 kDA