For how long did you incubate your zymography an din which buffer?

16 hours (cacl2, tris and zncl2)

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% Gelatine final concentratio? Is my gelatin bad

0.1% means 1/1000. Thus your final concentration should be 1mg/mL. I think you mess up your dilutions. Good luck

Hello Sulai, for zymograms with 10% of acrylamide an gelatin 0.2%, we weigh 0.036 g and dissolve them unto 10 ml of bidestillated water. Maybe is a gelatin concentration problem

Hello, I have another question for prep. of sample before loading on gel...

Your samples are native or denature condition (heat treatment or containing inhibitor in sample such as EDTA or EGTA).....

I checked my gel I guess I used too much of gelatin!!

No, I didn’t do heat treatment or had inhibitors in samples.

Hi, The percentage of the gel better be 10% or more to separate MMP-2/9 and their pro-enzyme, active forms. Don't use less percent gels. We can see in your picture the staining of proteins but not gelatin lysis. Try to use lysis buffer/sample buffer in non reducing conditions and load on to gel with out heating after incubating in sample buffer for some time(5-15min).

Thank you. We prepared 8% gel but used 1% gelatin final concentration. People recommend 0.1 or 0.2.

In my experience, 0.1% gelatin is ok and I run a 10 to 12% gel. Good luck

Thank you. I messed up with gelatin concentration. will try again. And may be increase incubation time too.

Dear Researchers, I optimized my gelatin conc (0.1 percent) and incubation time (40 hr). The following results i got. How shall I locate mmp2 and mmp9 in my gel? Is it good enough or it needs further optimisation?

Many thanks.

I think that in order to get clearer MMP9 bands you should aim for 0,2% gelatin, incubate a bit longer, stain with a strong and fresh (filtered!) Coomassie as long as it's very dark (1-2hr or something) and destain long enough to see the bands. It's hard to determine where it is, because the standard it not very visible, maybe you should put a bit more. There seems to be a problem with the sample itself, because it looks a bit degraded though. I never did cardiac samples and don't know how much MMP9 they have. It's also advisable, if you have the possibility, to scan wet gels (in a plastic sheet of course) with a transparent light scanner, because it really brings out the bands.

Thank you for your valuable suggestions.

My bad, I just checked I prepared 0.2% gelatin. I followed this protocol:

http://www3.szote.u-szeged.hu/hurodocs/downloads/biochemistry/HURO_zymo_practical_Kupai_final.pdf

The protocol used 8% separating gel. Is it too low do I need 10%?

Well, I stained less I guess (1 hr & 20 min). I shall troubleshoot this too.

The staining time is not as relevant as the color you see, because it depends on the quality of your Coomassie. If it's used several times, it starts to get diluted. It should be nicely, uniformly dark before the destaining (it's true that MMP2 bands are often visible at this stage, but not always). 8% or 10% should not matter. The best bands are with 1mm spacers. It is good to prepare gels the previous day and keep it in the fridge, wrapped in prewetted paper towel and foil with a comb still inside, it makes them prettier when they run. This gel looks a little bit like it could use longer polymerization (or fresh APS...). The dissolved gelatin stocks - we keep it in aliquots in -20 or -80, no need to make fresh each time, just warm it well before use.

If the quantity of MMP9 in your sample is low, and you don't need MMP2 (which is already oversaturated), increase your sample size.

You optimize your method to make it look pretty. This is what you're after: pretty, even, not oversaturated bands. Then you record your run times, incubation times, staining times etc. and repeat it every time the same way.

And always put in the standard :D

Yes, I did observe mmp2 bands even before destaining. Unfortunately, we have glass plate with only 0.75mm spacers. We prepare fresh APS. My samples are post myocardial infarction and I am not expecting low mmp9 activity at least in my diseased group. I would like to store gelatin in -20. But I actually prepared my gelatin with sds and distil water. Warming up gelatin can destroy sds or cause something bad?

I would like to know about storing gel a day before running. You keep the gel within the plates and wrap it so? What about the casting frame? Can it be removed once gel is polymerised?

I store the gel within the plates, with comb and spacers (Biorad has plates with spacers, so it's even better). I wrap them in pre-wetted tissue paper (distilled H2O) and then plastic foil such as used for putting paper into binders, or other sealed plastic bag. Once it polymerizes you don't need casting frame of course. You take it off just like before you assemble the apparatus to run the gel.

SDS is not destroyed by warming. After all it's in your samples too :) I don't remember what temperature i was defreezing gelatin stock, 37 or 50, but I think both is ok. It should be well dissolved.

Thank you so much!!

56°c is ideal if you want to defreeze gelatin. At this temperature, you also denature all the collagens that may still be in your solution. 2 birds with one stone.

Greetings everyone! I did some troubleshootin. Firstly, I prepared gels a night before my run. Secondly, I loaded 60ug protein (3 folds lesser than previously I loaded). The bands look prettier than before? MMP 9 is so low that when I take it into image j I don’t get any curves for the band. I kept a positive control too which my colleagues used for cell culture experiment. Any more suggestions?

i cant scan good! I don’t know what is the problem. The gel is not that bad but these captures are not doing justic.