I am having a really tough time with a flourescent reverse zymography
I work with a protease inhibitor (TIMP), and so I run reverse zymograms to
test for inhibitory activity. The problems with reverse zymography are that
you can't tell if the gels have incubated long enough (ie you need a
real-time way of checking them and carry on incubating if required, before
fixing and staining the gels), and whether the bands that come up with the
stain are actually bands of inhibition or simply overloaded protein bands
that would come up anyway with Coomassie.
The solution to these problems (or so I thought) would be a flourescently
tagged zymogram (I've used FITC-labeled gelatin). With this, I could stick
my gel under UV to see if it was done, and if not, put it back in the
incubator until it was. Then, I could take a photo of the UV bands, as only
the bands of undigested FITC-gelatin should show up (ie no extraneous
protein bands showing up with Coomassie).
Ha, Murphy said, as he laughed at my attempts!!
The problem is that the contrast between the background FITC-gelatin and the
undigested FITC-gelatin is so high I can't get a decent picture. I have
tried diluting my FITC-gelatin with normal gelatin to "tone it down"
somewhat, but then the fluorescent bands I want are so much weaker too.
Ideally there should be no background - it should all have been digested
away, but I've tried increasing the amount of protease co-polymerised in the
gel, with no difference either.
Has anyone done anything similar - any ideas or tips/hints/suggestions would
be greatly appreciated!