Hello Sulail! Yes, I store the gelatin in a normal fridge, I prepare it, heat it until dissolution and then it usually stays dissolved. If not, I just heat it prior to use again. You may want to aliquot the solution, and each time you prepare a gel you use an aliquot.

Regarding Triton, I would not use it multiple times, since you may have enzymatic contamination and ruin your gel with Triton from previous incubations/washings. I use only once and discard.

Thank you for the answer. Is there any alternative for triton X 100? Like tween-20? If tween 20 is good, shall it be prepared same 2.5% v/v?

This thread says its ok: https://www.researchgate.net/post/Does_anyone_have_a_suggestion_to_make_the_renaturation_buffer_for_zymogram

but then someone says it´s not a good resolution at the end of the process... Your choice!

Please go through this post too.

https://www.researchgate.net/post/Why_no_bands_in_gelatin_zymogram_And_something_unusual_at_the_bottom_of_gel

Are these mini gels? How much total protein are you applying? You should be seeing something much more visible, like so:

No they are not mini gels I loaded 80ug and 100ug. No, they are not mini gels.

Have your samples been thawed and frozen over 5 times? You should be seeing something much better than that of your gels, much better defined bands... is the final gelatin concentration 0.1%?

I prepared gelatin solution as 1g in 10 ml (that is 10%) and used 1ml for running my gel which had total volume of 20ml.

What is meant by 0.1%?

No, my samples were thawed not more than twice.

Hi,

I would like to see your protocol.

I think one wash with 2.5% triton X-100 for 30-60 min is sufficient to remove SDS...you don't have to give multiple washes, but yes you can give quick 2-3 washes but not with the used buffer.

In my experience freshly dissolved gelatin at 37oC gives good resolution and sharp bands. I generally use 1% gelatin for zymogram. Please go through this protocol:

"To assess gelatinolytic activity, test samples were mixed with non-reducing sample buffer and run on SDS-PAGE (10% or 12%) wherein the resolving gel was copolymerized with gelatin (final concentration 1 mg/ml) to function as a substrate for gelatinases. The gels were carefully removed and incubated in renaturing buffer (2.5% (vol/vol) Triton X-100) for 30 min at room temperature. The renaturing buffer was removed and replaced with developing buffer (40 mM Tris–HCl, 200 mM NaCl, 5 mM CaCl2, 0.02% (vol/vol) Brij35; pH 7.5) for a further 30 min. Fresh developing buffer was added and gels were incubated overnight at 37 °C. The gels were then stained with Coomassie Blue; gelatinolytic activity appeared as clear bands on a blue background."

Good luck

Hi again. I adjusted my gelatin and got something but mmp9 is not clear. I followed Krisztina Kupai‘s protocol but I washed twice with triton -x and incubated my gel for 40hrs in zymography buffer.

This is what I got.

The big veritically broad bands in the last lane was my positive control cell culture medium. I loaded 120ug of proteins in all wells. Can anyone suggest please how to troubleshoot for fine bands and more better mmp9 bands. This time it’s. MiniGel. 8% with 0.2%gelatin I used fresh gelatin.

You loaded too much protein! On mini gels I load 25 ug! Try this and get back to us! :)

Triton, like other polyoxyethylene detergents, tends to form reactive peroxides with air oxygen. These can seriously affect proteins. Buy the peroxide-free quality (in-house purification is possible, but tedious and requires nasty chemicals), store cool, protected from light and under N2, and use any solutions speedily.

Greetings everyone,

I got this with 50ug. It looks better? I am not happy with my mmp 9. It’s not that visible. For some samples image j is not detecting bands. Rachel, can you share your reference? Your gel is 8 percent or 10?

Intrestingly, tried tween 20 as my triton x is coming to an end. The gel with 2 samples. It seems a nice alternative for triton x.

p.s. The capture is from my phone with a weird camera I guess the scan has to be better. Is there something to consider while scanning?

Much better, but have you thought of the possinility that your samples simply do not express MMP9? I see faint bands above and below the strong ones. What are the MW of the standards? The strong ones should be MMP2, right? Can´t you work with that? When you conduct the densitometry analysis you will be ale to quantify the pixels in the bands, even though they are very faint.

Yes, strong ones are mmp2. Well, it’s not possible to have low or none mmp9 as literature emphasizes it’s high activity at least in my diseased group. I am planning to increase my incubation time to like 50 or 60 hrs for next trial. Can you share your precise protocol, please?

I incubate overnight only, my protocol is in portuguese, sorry hehe

Well, basically I run the gel, renature with Triton 4 x 15 min and then incubate overnight at 37o

I use the Troeberg and Nagase protocol

Lol. thanks for the help though. I washed my mini gel with 100ml only once. I can try your step.

Hi Have you tried the protocol of Rachel?

The final concentration of your gelatin was of 0.2%?