Hi,
I am performing gelatin zymography to see MMP 2 and 9 expression. I am having several technical difficulties. I am using conditioned media from 3T3-L1 preadipocyte and MDA-MB 231 cells. I am using Novex zymogram reagent and Invitrogen gelatin precast gels for zymography.
I am using FBS as a control. My bands are not very clear-any protocols in using conditioned media?
Also the gel breaks even when I run it on a low voltage-any tips?
I am also having trouble visualizing the gel using the GeneSys machine.
Any tips, protocols, etc. will be great! Thanks!
I am using the protocol here :http://protocol-place.com/assays/gelatin-zymography/gelatin-zymography-protocol/
Hi Danielle,
You can check in my article the protocol that we used to perform gelatin zymographies using homemade gels.
Article Stimulation of cell invasion by the Golgi Ion Channel GAAP/T...
Here are some tips that I know of,
1. Use positive MMP controls to see if it's a concentration problem (if MMPs in your medium are dilute)
2. If they are dilute, concentrate your sample using filter (30kDa) and then load your samples.
3. Allow more time for bands to separate.
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