detection of a secreted recombinant protein - (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 or has any suggestions on other ways to determine if a recombinant protein is being secreted.

In case this helps, I used DMEM + 10% FBS to culture smooth muscle 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!

---

Have you check if you considered dilution factor? if you want to stick with WB, you may need to dialyse/concentrate your supernantant, reconstitute it the same volume as you used for cell lysis to make a fair comparison.

Of course you can also use ELISA to quntitate how much it is.

---

Thanks for your help.

I have tried to concentrate my protein using a membrane/spin procedure, but was unable to detect protein. I will try again using the TCA protein purification method. Unfortunately there is no available ELISA procedure, but perhaps I can develop one using the tag I added to my protein. thanks again.

---