“Activity 5” Which Goes With Lab 4
The Kaleidoscope standard mixture that you ran in lab 4 is composed of various molecular weight proteins. You will use their known molecular weights to calibrate your gel, and then use the calibration to determine the molecular weight of your denatured proteins.
1. Open Excel and type in column A the molecular masses of the Kaleidoscope standards in kilodaltons (kD) in descending order. The names are not important at this point.
2. In column B, type in the migration distances of the spots in the Kaleidoscope standards lane of your gel. These distances should be measured in millimeters (mm) from the bottom of the standards well to the middle of the each spot in the standards lane. This is why a good photograph of the gel is so helpful. Type these in ascending order.
3. If there are any extra rows, assume that the smallest protein(s) have run off the bottom of the gel and therefore are not represented by any spot on the gel. Go ahead and eliminate those “unmatched” rows, using the delete row command in Excel.
4. Use the scatter graph function on Chart Wizard to plot molecular weight of protein in kD (x-axis) versus migration distance in mm (y-axis). Type in an appropriate title and don’t forget to add the axis labels.
5. Is this a linear graph?
6. Now use the add column function to open a new column B between your existing two columns (the old column B will become the new column C). In the new column, go to the cell next to the first data cell in column A and type “=log(“, then click the first data cell, then type”)”. Your completed cell entry for the B column cell should be “=log(A2)” or whatever A column cell your datum was in. Press return, and there should be a number in that cell that represents the logarithm of the number in the A column cell.
7. Click and drag from that cell down column B (except for the first cell, these should all be empty) until the end of data entries in column A. For instance, if you have data until cell A7, then you should stop dragging at B7.
8. Use the command Edit à Fill à Down to fill this column with logarithms of the entries in column A.
9. Now plot the logarithm of molecular weight of protein (x-axis) versus the migration distance in mm (y-axis). Continue to use scatter graphs, and add a title and appropriate axis labels to each axis.
10. Is this a linear graph?
11. Finally, use the same logarithm-producing procedure to generate the logarithms of the migration distance column.
12. Plot the logarithm of molecular weight of protein (x-axis) versus the logarithm of migration distance in mm (y-axis). Continue to use scatter graphs, and add a title and appropriate axis labels to each axis.
13. Is this a linear graph?
14. Choose the best linear graph of the three and draw a best fit line through the data points, by using the Chart à Add Trendline function (or else drawing it in yourself).
15. At last, use Chart à Add Trendline à Options à Display Equation on Chart (or else eyeball it) to convert the measurement of the migration distance of the fish muscle proteins from their wells to their spots in their respective lanes into actual molecular weights in kD. Recall that if you have a logarithmic measurement, you will have to perform an exponential (or anti-logarithmic) function in order to get a sensible answer. In other words, the molecular weight of a protein (kD) = 10(migration distance (mm)).
You don’t have to do the exponential if you obtain the log-log graph paper.
16. For your postlab in your lab report, attach the Excel data table(s), the graph(s), and molecular weights for all proteins in your gel.