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Learning Module - Experiment 11B

Densitometry of SDS-PAGE Gels and Western Blots

Use the "ImageJ" program to generate "densitometer scans" and obtain the integrated-area data for each lane on your Coomassie Blue stained gel as described in the coursepack.

For each peak in the MW size standards, calculate a conversion factor (integrated area / μg protein) for each resolved peak, and then calculate the mean and standard deviation. This yields a factor to convert the integrated area of "other proteins" into µg protein.

Calculate the conversion factor for Topo I, keeping in mind the mass of pure Topo I that you loaded on your gel.

Analyze the data for the lysates from induced and uninduced cells:

Estimate the amount of protein in the Topo I peak in each lysate as well as the amount of protein in the "other proteins" peaks. Use the appropriate conversion factor for Topo I estimation, and the conversion factor from the size standards for the other proteins. Then estimate the percent of total protein that is Topo I in the two lysates.

Finally, compare the percent of protein that is Topo I in the +IPTG and -IPTG lysates and estimate the -fold overproduction.

This estimate assumes that the peak we are calling Topo I in the -IPTG lysate is all due to Topo I and not some other bacterial protein that happens to be the same size as Topo I. In order to quantify accurately the amount of Topo I present, we need to use a method that is specific for Topo I. The immunochemical detection method is ideally suited to this purpose. Compare the results you obtain by using densitometry of your Western blot to that obtained with the Coomassie blue stained gel.

Analyze your integrated-area data from the Western blot in a similar manner to that used for the Coomassie-stained gel:

First determine a conversion factor for pure Topoisomerase I, remembering that the sample contains 400 ng of Topo I.

Then move on to your lysate samples and calculate the amount of Topo I in each lane. For each lane, calculate the % of total protein that is Topo I, based on your estimates of the amount of Topo I in the lane, and the known amount of total protein loaded in each lane. Finally, estimate the -fold overproduction of Topo I. For the most accurate estimate, you should compare the lanes from the lysates of induced (+ IPTG) and uninduced (- IPTG) cells that have amounts of Topo I that are nearest to the amount in the pure Topo I lane

Compare the results of the different methods you have used to quantify the amount of Topoisomerase I in your lysates and the -fold overproduction. Why do the different methods yield different values for the level of over-production?