I have to analyzed the expression of MMP-2/9 from cell soup contained medium.
If I understand correctly, you want to measure MMP-2 and -9 in the conditioned media of your cells.
If that is true, then you usually wouldn't quantify protein. Just load conditioned media by volume (for example, 10 ul per well).
But make sure that you didn't add serum (FBS, FCS, etc.) to your cell culture media. Serum contains a lot of MMP-2 and MMP-9, and you will end up measuring that instead of the MMPs that are being released from your cells.
you should load the same number of mg of protein per well. Also, you may need to concentrate the media to see the activity, and total protein loaded to each well should be determined.
Sure, you have to measure total protein contents and load equial amount, In addiotion, you can also use additional normalization against well-know protein to be sure that your calculation is correct.
Taras, which well-known protein would you recommend for normalization? Also, would you quantify this protein after staining of the gel (with Coomassie for example)?. Thank you.
Well-know protein? Like housekeeping protein? As far as I know, there is no well-known protein in conditioned media that could be used. For cell extract, we could, e.g., actin.
For cell soup protein analysis you need to do ponceau staining @Amira Zine EI Abidine. Hope this will help you.
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