Thank you, Nando!

Yes, I want a detailed protocol for gelatin zymography (for MMP-2 and MMP-9). And the paper you’ve sent is exactly what I needed. Thank you once more. But I have a few questions.

1 What for did you take 2 detergents (Brij®-35 and Triton X-100) in one buffer?

2 As for inhibition studies, inhibitors should be added in the enzyme buffer at the beginning of 24 h incubation, isn’t it?

All the best,

Tania

Thank You, Nando!!!

Hi, Nando!

Can I ask you one more question?

Which molecular mass markers do you use for zymography?

As for zymography, it goes perfectly well. Thank you ones more.

We run a heavy load of standard pre-stained MW markers, which can be seen even with the gelatin background. We also frequently run a lane with a known amount of either human recombinant MMP-2 or MMP-9 in one lane as a standard.

Thank You, Bryan!

If you run standard in one lane, you may skip using molecular weight marker. That will be economic !

In case of gelatinases, we do not use Brij35, 2 washing with Triton X-100 is sufficient to replace the SDS and to renature the matrix metalloproteinases. But, in case of caesin zymography, we do use Brij 35 in incubation buffer.

It would be very nice to have a standard, but we can't afford it. I'm working with chikhen mmps, and it is difficult to distinguish between mmp-2 and mmp-9 as long as thay have very close molecular weights.

Thank you for tips for Brij35.

MMP-9 and MMP-2 are not close in molecular wts. MMP-9 is 92 Kd in the pro form and about 2 KD in active form, whereas MMP-2 is 72 in pro form and 62 in active form.

It is easily distinguishable.

When speaking about human MMPs, yes, they are easily distinguishable. But chicken MMPs, I'm working with, are 75 kD (MMP-9 like gelatinase of chicken), 72 and 62 kD (pro- and active form of MMP-2 respectively). The molecular weight marker which we used to use is invisible (when is loaded as recommended) or looks like a smile (when overloaded), so I can't distinguish whether it is 75 kD MMP-9 or 72 kD MMP-2. So, I'm wondering what is better and chipper: to buy two standards of chicken MMPs or to buy a good mol.weight marker. I've just started to work with MMP, so I'm seeking advice.

That is tricky. Are there any antibodies that cross-react with chicken MMP-9 or 2? You might be able to run your gels in duplicate and figure out what's what by western blotting one and doing zymography with the other.

I'd recommend trying fairly high percentage gels (12 or 13%), and running them slowly for a long time (use prestained MW markers and run until the 75 kD band is near the bottom of the gel). That should get you enough resolution that you'll be able to distinguish 75, 72 and 62 kD.

Thank you both for help! Overunning is worth trying.

Bryan, could you tell me how your mol.weight marckers are called?

Tania! could you please provide me the protocol for the Gelatin zymography. I m working on MMPs. I m new for Gelatin Zymography. But above comments are helpful if I have std. using/successed protcol... Thank you in advance.([email protected])

1) I have very low expression of MMPs in my cell line i.e., conc. of MMPs is very low in total protein I have taken.

2) How much gel % I need to use if its resolving?

Hi, Chandra!

The very first comment in this discussion by Ferdinando Mannello has the reference to his protocol of zymography. I’m sending another one to your email. Good luck!

thank you,Tania!

does any one has idea that do DU 145 or LNCaP cells produce MMPs in detectable amount. If not does any way to increase its expression by changing media composition.?

If you want to assess Multiple MMP activities in addition to 9 and 2, there are FRET protocols for this ( fluorescent substrates).

Hello,

Tania, I'm also new in gelatin zymography. Would you sent me also that protocol at [email protected] ?

Thank you!

Hello,

Can i also get a good protocol for mmp-9 detection gelatin zymography and western blot (rat tissue, small tissue).

Would you sent me at [email protected]? 

Thank you in advance.

"Assessment of Gelatinases (MMP-2 and MMP-9) by Gelatin Zymography" by Marta Toth and Rafael Fridman in Methods Mol Med. 2001; 57: 10.1385/1-59259-136-1:163. 

See attached.

hi Anirban, can we use Triton x -100 in place of Brij-35

Hi, Rashmi Shukla!

Triton X-100 works pretty well

thanks, Tania.

you are welcome :)

Hi tania,

i am not getting proper bands. m sending you zymography gel picture.      

Hi, Rashmi.

Maybe, the zymograhpy was running too fast or it was too hot in the experimental room.

Hi, Edita!

Thank You for the article you've sent me. It is excellent!

Dear Tania and all researcher in this group

Can you send me the catalogue no. of ''Gelatin'' and ''beads'' please?

I am ordering stuff for Zymography  and need advice from expert already done the experiment. I am in very critical time and need help please....

Regards

Dear Tania and all the researchers in this group,

I am currently doing gelatin zymopgraphy as well and thank you very much for all the information above. My lab has basically everything listed in the articles, except for Brij 35. 

Some of you have suggested to use Triton X-100 to substitute Brij 35. But I am not sure which of the following is the correct way of doing it:

a) Using only Triton X-100 as the developing buffer, or

b) Using Triton X-100 to replace only Brij 35 as part of the developing buffer?

If a), then is the incubation time still the same as suggested in the article? 

Thank you very much.

Hi, Dylan Lim!

I prepare all the buffers as describe in the following article, but simply do not add Brij 35. And they work good.

"Assessment of Gelatinases (MMP-2 and MMP-9) by Gelatin Zymography" by Marta Toth and Rafael Fridman in Methods Mol Med. 2001; 57: 10.1385/1-59259-136-1:163.

Hi, Morteta H AL-Medhtiy!

Unfortunately, I can't help you, as long as I use Gelatin of local Ukrainian producer.

Dear Tania

Thank you so much for your answer...

I'd like to inform any one who looking for that issue, the author answered me and the link for Brij 35 is the following:

http://www.sigmaaldrich.com/catalog/product/sial/b4184?lang=en®ion=GB

Regards 

To clarify: YES triton-x can be used as a substitute for Brij in may instances. The detergent is added to the Gel Washing Buffer. To make GWB:  add 20 ml Triton X-100 + 980 ml of di H2O. Do not take short cuts in the gel washing step. Wash the gel 3 x with 50 mL gel washing buffer for 15 min each time. Incomplete wash will limit collagen degradation in subsequent steps.

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