Respected Sir,
I am a research scholar at department of zoology division of developmental
physiology at M.S.University Baroda. I am working on lizard tail
regeneration . and i need to find out the role of MMP-2 and MMP-9 in it. so
i performed gelatin zymography. i performed the protocol as follows....
- i prepared the tissue homogenates in non reducing medium.
- preparation of stacking 4% and separating 10% with 0.5 mg/ml of
gelatin( sigma type A porcine skin electrophoretic grade) in it
- then washed with 2.5% triton x 100 for one hour and two changes
- then incubated in incubation buffer for over night it consist of
CaCl2, NaN3, Tris HCl Ph 7.5
- after that gel is stained with coomasine brilliant blue for 12
hours.
- and lastly destained with aceto-methanol....
but the problem is when destained i do get some bands but they are not
transparent n also the background of the gel is not blue after destaing
it.....as many papers i had sen its written in the blue background you will
see bands....
so where i am wrong in the protocol and what should i do . to make it
successful
your great help will be very important in my work and i hope to see your
reply back
kind regards
Priyanka Sharma
