I am treating cells with cathepsin D. I would like to confirm that cathepsin D in this case is acting in a proteolytic-independent manner. As the pH of the culture media goes down to 6.8, there might be a slight chance of cathepsin D acquiring its proteolytic activity. I have heard of fluorogenic substrate for cathepsin D, but I don't know if that can be used cell culture media or how. People have previously used it in cell lysate to test intracellular/lysosomal cathepsin D activity. Any suggestions on how to test externally added cathepsin D proteolytic activity in cell culture please?
You can use a fluorimetric assay to determine the activity of Cathepsin D in the cell conditioned medium. I have tried one of these kits and it works really well. here is the link: http://www.anaspec.com/products/product.asp?id=53166.
You can probably also find it from other suppliers if you want to shop around. but the basic principle is using a fluorogenic substrate.
i was able to detect activity of another cathepsin, using a similar kit, from conditioned media of cells cultured in 48-well plates. I collected medium at different time points, and all you need is I believe 50ul of your medium.
Good luck
That is excellent! I have come across this fluorimentric kit and have planned accordingly. Thanks a lot Tina!
Cheers Karima!
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