Learning Module -
Period 20
Densitometry of
SDS-PAGE Gels and Western Blots
Use the "ScionImage" 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?
Procede to LON-CAPA to answer the
question for this Learning Module.