Do you have a protocol to concentrate media for detecting secreted proteins like MMPs?

We use Microcon columns from Amicon (30 kDa cut off) to concentrate media. It is important that the medium does not contain FBS because this will make it difficult to concentrate with the Microcon columns.
1. Spin the media (20 ml) to pellet any cellular debris.
2. Add the 20ml volume to a Microcon column and concentrate it down to 500 ul.
3. Add 10 ml of cold 1xPBS to the 500 ul sample to wash it and concentrate again to 500 ul.
4. Transfer the 500 ul of concentrated media to an eppendorf tube and wash the Microcon tube membrane twice with 250 ul of cold 1xPBS to remove any proteins stuck to the membrane. This should result in a 1ml sample.
5. Add 500 ul of 3x reducing SDS sample buffer to 1.0 ml of concentrated media. MMPs tend to be quite unstable and degrade easily in our hands, so we highly suggest adding protease and phosphatase inhibitors to the lysate, along with PMSF and 5 mM EDTA (as a chelator).
6. Aliquot the lysates and store them at -80C.

 

Last updated: September 12, 2024