optimal timepoint to collect secreted protein - (Oct/23/2008 )

Hi all,

I'm having problems with obtaining my protein of interest so that i can assess it by western. The function of this novel gene has yet to be defined. I have used numerous protein function prediction programs and there is a likelihood that this protein is secreted.

My questionn is, if i transfect COS-7 cells with an expression construct (pcDNA3), when is the best time to collect protein if it is secreted? i have tried to collect protein 24h post-transfection from cell lysate, supernatant (by precipiation) and also treating cells with Brefeldin A and collecting the cell lysate but i have not been able to find any protein. My postitive control (a protein which is not secreted) is coming up ok.

Thanks for your help

I would suggest that you collect your protein in a time series, starting at 12 h or earlier (6h) after transfection and going on every 12h up to 72h or 96h. This will give you a good overview over the expression and secretion, especially important since you know nothing about it just now. I would do this in the beginning 3 time to be sure to get a good picture of the right time points and then reduce the procedure to the interesting ones where your protein is detectable. Kind of tedious, I know, but you will save a lot of time later.