I have tried to make new solutions required for gel casting (30% acrylamide/bis, Tris-HCl pH 8.8 and 6.8, casein 0.5%), sample buffer, the staining solution and destaining solution, because the gel staining was even and not patchy 2 months ago.
I also make fresh APS solution every time I am about to make the gel. the only solution I haven't made again is the running buffer.
could it be the cause?
the TEMED I use is also no longer good, the top part always won't solidify completely and makes jagged pattern. but I put a little isopropanol on it, it helps to make the gel solidify evenly.
could it be the cause too?
and also, should I make fresh casein solution every time?
thank you for any help in advance.
Hi Evelyn,
I've done a few of these zymograms over the years and have also found that I got "patchy" staining occasionally. I found that freshly prepared buffers (including the casein) and fresh APS are essential. Your TEMED is probably ok...I never had a problem with that. The other thing to consider when making your own gels for zymograms is to make sure that your glass plates are really clean. I used to clean mine with chromic acid, which is hazardous stuff and I wouldn't recommend it to anyone today. A lab glassware decontaminating solution like "Decon 90" will work well. Soak for 60 min and then give the plates a good scrub. Lots of rinces with DI. water and a final wipe over with ethanol (some people also use acetone).
I hope this helps.
George
Dear colleague:
I had the same issue with gelatin zymography for mmp9. The problem was fixed when we prepared a new gelatin stock for each experiment. Also, the gelatin (in my case) must be diluted at apropiate temperature, because if the temperature is not adequate (too low), gelatin will not disolved completely, and therefore, we obtain gels just like yours.
I hope this little piece of advice can help you with your problem.
Wish you luck!!!!
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