secreted protein quantification - (Dec/08/2010 )

Hi, I really need help about the following...

I have 3 different proteins that are secreted in a cell culture media without serum. One of them is v5 tagged. The two other are not tagged but they contain the Fc region from the human IgG2b. My question is how to quantify the amount of protein contained in each media keeping in mind that I have to compare the 3 of them. I cannot use the anti v5 antibody in my Elisa protocol neither the anti protein A-HRP for the other two because I won t be able to compare the 3 of them. So please can you help me???do you know any method?? I don t need to have it very precise but only to be sure to put the same amount of them for my experiment. Thanks!

Hi!
Just thinking about your situation. I hope that my response doesn't sound too inane - I'm more of a DNA person ! Could you look into western for quantitation? Also, what about looking for other epitopes in/on your proteins? Perhaps there are antibodies that correspond to each secreted protein specifically? Or is it possible to look into developing specific antibodies to your secreted proteins? Anyways, Good luck with your research!
~Nic~

Hola, If i were you I would precipitate same amounts of medium (100-200 ul)with 10% TCA and made a PAGE of the suspended and neutralized pellets applying known amounts of a protein with similar MW (in other wells) staining with Coomassie blue and comparing intensity visually or densitometering. Buena suerte