determining if a recombinant protein is being secreted - (Jul/18/2006 )

Hello,

I am trying to detect a recombinant ECM protein in the conditioned media to determine if it is being secreted. So far I have had no luck. I was wondering if anyone has any experience with this and has any suggestions on how to determine if a recombinant protein is being secreted.

In case this helps, I used DMEM + 10% FBS to culture my cells, collected the media, spun it down to get rid of cell debris, and then ran a standard SDS/PAGE, transfered it to a membrane and detected with my Ab's. I am getting a strong signal from the lysate but no signal from the conditioned media.

Thanks for your help!

Hi,

When you say you spinned down the debris, did u concentrate the media itself before loading to the gel?? If you havent done that might be you need to concentrate it down and check it out, might be the protein is secreted in very low concentration.

Secondly might be you should check out the secretory signal sequence being added properly, as you are getting the protein in lysate there might be chances that secretory sequence is not taken up!!

May b its not being secreted (due to some unknown reason), thats y u get a good signal from lysate (the protien is still within the cell).

have u tried to detect with ELISA.

I would recommend concentrating the media as well, although with 10% serum you will not be able to reduce volume very much because of the high amounts of serum albumin in your sample. You can concentrate about 10-fold without problems in 1% serum, although that would also require using a different media formulation for you.