Zymography/Detecting pro-Zymogen - How does pro-enzyme digest the substrate? (Jun/05/2007 )
Hello everyone- I am new to this technique of zymography. I understand that a band for the pro-enzyme also shows up when stained with commosie blue stain. So, my question is - How can a pro-enzyme (thats not active) digest the substrate on the gel? Also, Are there any specific protein ladders for zymography? Thanks a lot in advance.
I have done zymography in this period. I'm not sure but I think that you can see the pro-enzYme form because first you run the gel: in the sample may you have both the active form and the pro-enzime form; then you incubate the gel to activate the protein: in this time proteins can activate one another and so remove the prodomain and then digest the substrate....But they can't diffuse in the gel so you will see the band at the molecular weight of the pro-enzyme form!
Then I suggest you a ladder for MMP2/MMP9: if you want you can buy it (see http://www.chemicon.com/browse/productdeta...roductID=CC073). As an alternative you can use HT1080 that is derived from human fibrosarcoma cells that overexpresses MMP2 and MMP9.
I hope these informations will be useful for you
I'm not an expert in zymography but someone who is explained it me like that:
proenzyme is active in the gel because, as you work with SDS-PAGE, it is denatured. So, the pro-domain doesn't inhibit anymore the catalytic domain which can therefore actively digest substrate. that's why the assumed inactive pro-enzyme can digest substrate.
I hope this may help.