Hi everyone!
I have the following problem:
I have a gfp tagged protein that localizes in damaged dna (i
microirradiate a part of the nucleus with a laser and my protein moves
there. In untreated cells the nucleus remain completely black).
I also have different mutants for this protein that seems to be affecting
how this protein behave (looks like the mutants enter the nucleus but don't
quite localize at the damaged sites like the WT protein).
I have different time points after the irradiation and i was approachibg
the situation like this:
1) Select a roi for the nucleus and another for the irradiated zone.
2) On the photos from the green channel:
-I do a threshold correction until the nucleus at time 0 is black.
-calculate the area and the integrated density for both rois.
- Move to the next time point. Apply the same value of threshold
obtained at time 0 and the do the same calculations.
-For each cell i calculate the intDen/area = Integrated density / area
for the entire nucleus, the irradiated zone and the non irradiated zone
(nucleus minus irradiated zone).
-Then calculate a ratio of the previous parameter versus the time 0 to
see the changes over time.
My idea was to compare how the fluorescence changes over time in the non
irradiated zone and the irradiated zones among the different mutants i have
but i'm not sure this is the right way to do it. I have a lot of cells and
mutants and before doing a lot of work i would like to know if this is the
right way to do it.
Many thanks in advance for your answers,
Gonzalo
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