Galactosidase on your gel?

SDS-PAGE Data Analysis & Discussion Questions

Part 1: Data Analysis (7 pts)
In this part of the assignment you will use the measurements of the protein migration distances that you recorded for your SDS-PAGE gel to calculate an approximate molecular weight (MW) for the protein you identified as -galactosidase. The instructions below describe how to use Microsoft Excel to generate a standard curve from the measured migration distances of the MW standard bands and then how to use the linear regression equation that specifies the graphed line to calculate the MW of any other band. You should submit your Excel spreadsheet as an attachment on Blackboard along with your answers to the discussion questions in Part 2 below; be sure to also show your work for the MW calculation.
Calculating the MW of -galactosidase based on your gel data:

1. Determine the migration distance into the gel of the MW standard proteins and the band identified as -galactosidase using a millimeter ruler.
2. Open a new worksheet in Microsoft Excel. Label the first column “Molecular Weight,” the second column “Migration Distance,” and the third column “Log Molecular Weight.”
3. In column A, record the molecular weights for all of the standard proteins. (Note: your instructor should provide you with these values.)
4. In column B, record the distance migrated by the each of the MW standards in centimeters or millimeters.
5. In the first cell below the label in column C (cell C2), type =LOG(A2,10) and hit Return. The log10 of the first molecular weight will be computed and placed in that cell. Click on cell C2 to highlight it, then drag the “handle” at the bottom right corner down to the last data row to compute the log values for the other measurements.
6. Highlight the labels and all of the data in columns B and C. Click on the “Charts” tab and select a scatter plot (may be in the “Other” category). A graph of your data should appear in the worksheet, with the migration distances on the x-axis and the logMW values on the y-axis.
7. Click on one of the data points on the graph to highlight the data series. In the pull-down “Chart” menu at the top of the screen, select “Add trendline.” Keep the default, which will be a linear regression. Under “Options,” click the boxes for “Display equation on chart” and “Display R2 value on chart.” The mathematical equation describing the best-fit line and the R2 (“goodness of fit”) value will appear on your graph. An R2 value of 0.95 or greater will represent high-quality (linear) data.
8. The general equation of a straight line is: y = mx + b
   where m is the slope and b is the y-intercept. In this case, the equation becomes: log10MW = (slope)(migration distance) + y-intercept
9. Determining the size of any other protein band (e.g. -galactosidase) becomes straightforward simply by placing the protein migration distance (x) into the equation and calculating y (log molecular weight). You can then calculate the actual MW using the relationship between logs and powers: 10^logX = X. In Excel you can use the formula =10^cell (where “cell” is the address of the spreadsheet cell that contains the logMW value).

Part 2: Discussion Questions (8 pts)

1. (2 pts) How did you go about identifying the band corresponding to -galactosidase on your gel? Describe the features of the data that allowed you to make this conclusion and explain your reasoning.
2. (2 pts) Can you tell whether there was any -galactosidase expressed in the t0 (uninduced) cells? Explain briefly.
3. (2 pts) Does your calculated MW for -galactosidase correspond to that of the monomer or the active tetramer? Explain why this is the case.
4. (2 pts) SDS-PAGE is also referred to as denaturing PAGE because the proteins analyzed are in a denatured state. Another type of protein separation by gel electrophoresis is native PAGE, in which proteins are maintained in their natural, folded state and migrate through the gel based on their net charges as well as their sizes. How would you expect your results to be different if you had subjected your bacterial lysates to native PAGE rather than denaturing PAG

Sample Solution