I wish to perform zymography for MMP-2 and MMP-9. I am working on cardiac remodeling and I have cardiac tissues as my sample to work on.
I am confused if I shall extract protein from my tissue or I shall isolate fibroblast and use the conditioned media or I shall simply perform in-situ zymography? Which way can give me best and reliable results.
I will be glad if someone can help me in this regards.
P.s. I can't afford a very high-budget work.
Thanks in Advance :)
Dear Sulail.
In response to your points:
a/ extract from tissue - give you a view of what is in your tissue, but complex to interpret, as it is at a specific single point, in a mix of cells. MMPs are also secreted and activated in the extracellular environment, so you have to think what a homogenate is telling you about this functional activity - e.g. Is the MMP stored in the cell, or in the ECM? this also means you have a more challenging cocktail of proteins on which to perform your zymography to work out what is what.
b/ conditioned media - can be done short term on explants, reflecting the in vivo state at the time of collection, and in cells - MMPs are secreted so it gives a view of the tissue MMP function / activity, and also permits an in vitro treatment. It is also a more simple sample to deal with and interpret.
c/ in situ zymography - it can provide insight into where any MMP activity is localised, and can be done in parallel with immunohistochemistry to localise the MMP protein. It can be technically challenging to get it to work consistently to allow you to make comparisons.
Finally - Gelatin zymography can be cheap. Make your own gels, degas and use them fresh, and you will find them highly consistent.
I hope that is helpful. Good luck.
Dear sulail
in response to your question
If you are interested in finding gelatinolytic activity of secreted MMPs then you can opt conditioned media (concentrate 10x and load). otherwise follow the given link
hope it will be enough.
http://www3.szote.u-szeged.hu/hurodocs/downloads/biochemistry/HURO_zymo_practical_Kupai_final.pdf
yes, Nem is correct. For conditioned media you will likely have to concentrate. You can use ultra amicon centrifugation concentrators from millepore. You can also use Fret substrates if you have access to a fluorometer. The substrates are selective and you can use minus and plus selective inhibitors. Biozyme sells the substrates. And Calbiochem sells the inhibitors. With this technique there is no need to concentrate the media. And you can use tissue lysates, or media from explants or cells.
I AGREE With Dr. Dulail, infact it is more important to estimate the status of mmps , whether it is in active or pro- form. Also in condition medial avoid serum as it will interfere with your result. Tissue too can be used but it is quite complex to impret. Generally activated gelatibases are of prime imporyance, that is mainly present in condition media. You can see protocols from any paper.
Good luck
Thanks every one :)
I had few concerns like. According to the papers I have read gelatinase mmps are in inactive form intracellularly and after binding to their substrates in matrix + proteolytic cleavage they become activated.
So if I take in account to extract mmps from tissue can I asses the active gelatinase activity using gelatinase zymography or my results will because of yet-to-be-activated forms too.
If I choose the media option can cardiac fibroblasts grow without expensive growth factors (as staying in the limit of my budget is important?) I came across a paper indicating FBS, DMEM, EMEM (+ antibiotics and collagenases).
you are right regarding the activation status of mmps. While preparing condition media see that the cells are almost confluent....this gives strength to cells for survival by self secreting growth factors.
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