Gelatin Zymography

Thank you BlueDevil...
My email id is bhakti.sadarani@gmail.com
Please send me the pdf file.....

Hii...i am trying to standardize the procedure for gelatin zymography. i am still a fresher to it. i have tried 5-6 times but i have failed to get any clear bands in my gel. I would appreciate if anyone could send me a standard protocol.
I wanted to know about the total protein load that should be made in the gel for the bands. Does higher voltage of the electrophoresis run cause any degradation of the enzyme? I am trying to detect MMPs in lung tissue homogenates and bronchoalveolar lavage samples. I wish to know for how long can the samples be stored at -76 degree celsius for getting the exnzyme activity? Also what homogenisation buffer should be used for preparing lung homogenates and what concentration homogenates should be prepared?
Thanks.

There is an excellent article available from Methods in Molecular Medicine, vol. 57:
Metastasis Research Protocols, Vol. 1: Analysis of Cells and Tissues. The chapter is Assessment of Gelatinases (MMP-2 and MMP-9) by Gelatin Zymography, by Marta Toth and Rafael Fridman.

If you post you email address, I will send you the pdf.

Hii...i am trying to standardize the procedure for gelatin zymography. i am still a fresher to it. i have tried 5-6 times but i have failed to get any clear bands in my gel. I would appreciate if anyone could send me a standard protocol.
I wanted to know about the total protein load that should be made in the gel for the bands. Does higher voltage of the electrophoresis run cause any degradation of the enzyme? I am trying to detect MMPs in lung tissue homogenates and bronchoalveolar lavage samples. I wish to know for how long can the samples be stored at -76 degree celsius for getting the exnzyme activity? Also what homogenisation buffer should be used for preparing lung homogenates and what concentration homogenates should be prepared?

Thanks.

Using gelatin zymography allows you to test for MMP activity while a western blot would just tell you if the MMP is present (but not if it's active).

Hi All,
I did not had any luck with zymography and i tried about 12-15 times. All i could see is few non-gelatinolytic coomassie stained bands. Is this because of high amount of sample loading? I would greatly appreciate if anyone can help me in accomplishing this zymography experiment. Oh, one more thing. when i used trypsin as control that did show gelatinolytic band.
Please help me.
thanks in advance.
Chandra

I came across this protocol a while back, unfortunately, I have never done one of these myself.

http://www.chemicon.com/techsupp/Protocol/gelatinzymography.asp

Hope this helps.

I have followed the procedure for zymography, but the clear bands didn't appear... where could it be wrong?

Just to clarify, from what I have read, leupeptin should not inhibit your MMPs in the gelatin zymography assay? You just wondering what it is doing to case multiple bands?

Is it possible that leupeptin is inhibiting another protease in your system that is involved in processing the MMPs? In the reference below, it mentions a system where they see multiple forms of MMP2. This might explain why you are seeing larger bands, however, it doesn't really explain the presence of the smaller bands.

Is it possible the smaller bands are a result of incorrect processing by yet a another protease or maybe an autocatalytic event that only occurs when the correct protease is inhibited by leupeptin?

Do antibodies exist that would allow you to do a Western to test whether the four bands are just different forms of the same MMP? You could compare the MW weights with those observed from the gelatin zymography.

If so, you could see what happens when you add leupeptin and EDTA or combine leupeptin with another protease inhibitor. Under these conditions, are all four bands still present by Western blotting? If you only see one band with leupeptin and EDTA, maybe the MMP has autocatalytic activity when incorrectly processed.

reference: Functional Interplay between Type I Collagen and Cell Surface Matrix Metalloproteinase Activity. J Biol Chem. 2001 Jul 6;276(27):24833-42. Epub 2001 Apr 30.

let me know what you think.

Original Message
From: xcabeto
Date: Thursday, March 17, 2005 5:35:42 PM
I am looking for MMP-1,2 and 9.First we used a different set of protease inhibitors to see if there were MMP's and if has multiple protease activities. Leupeptin is a cysteine-containing proteases inhibitor. But since this particular bands show up we would like to know what could be the reasons. Related to our MMP of interest has been checked with the inhibition by EDTA (chelator).

Original Message
From: smartguy
Date: Thursday, March 17, 2005 5:21:45 PM
Which MMP are you looking for? Have you compared this to a zymogram of the MMP without inhibitor?

Many MMPs will have more than one band on a zymogram because they form dimers and degrade to truncated versions that are still active. You may need to incubate longer to inhibit the activity of your MMP completely.

Also, are you sure leupeptin inhibits the activity of the MMP you are looking for? I'm not certian that leupeptin acts on metalloproteinases.

Original Message
From: xcabeto
Date: Tuesday, March 15, 2005 10:22:07 AM
Hi:
When using leupeptin as an inhibitor for matrix metalloproteinases in a gelatin zymography, I obtained four(4) bands instead of one (uninhibited)or none(inhibition). Two bands above the expected position and two bands below the expected position. What could be the behavior of leupeptin in this case?

I don't have a ton of experience with gelatin zymography and MMPs, but let me know if this helps.

Do you see any difference with and without leupeptin?

If you don't add any leupeptin, do you see only one band? If not, is it possible you have multiple MMPs in your sample that vary in size and hence migration.

Is leupeptin known to inhibit MMPs?

After doing a google search, chemicon has some info about several MMPs that seem to give multiple bands, is it possible this is what you are seeing?

http://www.chemicon.com/Product/ProductDataSheet.asp?ProductItem=AG770

Original Message
From: xcabeto
Date: Tuesday, March 15, 2005 10:22:07 AM
Hi:
When using leupeptin as an inhibitor for matrix metalloproteinases in a gelatin zymography, I obtained four(4) bands instead of one (uninhibited)or none(inhibition). Two bands above the expected position and two bands below the expected position. What could be the behavior of leupeptin in this case?