We are trying to demonstrate the our treatment effects increases the activity of MMP-2 by showing mutiple cleaved fibronectin bands in a western blot? For this we are going to have a control of pure fibronectin (hopefully one band) and also a control of fibronectin cleaved with MMP-2. What I am wanting to know is if anyone has a protocol on the incubation of a protein with an MMP? How long, what temperature, what concentrations etc?
Thank you!
Hello
Kindly look at links here
Article Tam EM, Morrison CJ, Wu YI, Stack MS, Overall CMMembrane pro...
Article A Comparative Study of Fibronectin Cleavage by MMP-1, -3, -13, and -14
Good Luck
Hi Sophie Upson in general MMPs are not very efficient proteinases compared to serine proteinases.
An easier way to determine whether your treatment increases the activity of MMP-2 is to do gelatin zymography. If there is more activity or if there are more low MW MMP2 bands showing up in the treated samples (compared to the MMP-2 zymogen), then you can say that the treatment increases MMP-2 expression or facilitates its activation from the zymogen.
You could also do a fibronectin zymography.
Dear Sophie,
I prefer to perform gelatin zymography in serum free medium of treated cells. You can find the details here.
Article The effect of sodium butyrate and cisplatin on expression of...
If you have any questions send me a message.
Regards Alena
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