Trouble with S35 labeling of exogenously expressed proteins - (Jan/24/2006 )

Hi,
I am trying to do some pulse chase analysis and having troubles in labeling my exogenously expressed proteins in Hepa1 cells. I want to know how long after transfection should I wait for labelling? How long should I label and how much S35 should I use in a 6-well format cell culture?. Does anybody have good experience with this experiment. Help will be much appreciated...

Thanx in advance.

Hi,
Can you co transfect your protein of interest with GFP and monitor expression over a few days by microscopy to see how efficient the transfection is. From this experiment you can determine how long after the transfection you have to wait for maximal efficieny before labelling. When I do S35 pulse chases, I've found its best for my cells (IM MEFs) to wait 24hours after the transfection before labelling. The length of time you label for depends on the half life of the protein (which you obviously don't know if you're doing this experiment) try 1-2 hours as a starting point. I use 0.0525mCi in a 6cm dish.