stainig of cells for FACS - (Jul/29/2007 )

I need to see intraclullar protein by FACS. Can some one tell me the protocol to stain cell for FACS anlysis ?

People use PFA to fix cells and saponin for permeabiliztion.

1. What is the optimum conc. and time period of incubation?
2. Can we use Triton X 100 instead of saponin to fix cells?

thanks

Personaly I use a kit called Cytofix/Cytoperm, but it depends on what you're staining. For example if u're looking at phosphorylated protein you'll have to permealise your cells with methanol.
I dunno if your question include PFA but I use it with a final concentration of 4% with an incubation of 1 to 24h according if your culture is infected or not.

Thanks
I did not know there is kit available for staining. Protien which I want to stain is infact could be phospho protein and I am planning another experiment to prove that.
could you please tell me how I can look phosphorylated protein?

I was planning Staining to see the localization of this protein.
I have also used 4% PFA in past for 20 minutes. (Induced cells to make virus)
But never tried to permealize cell I need to permeabilize cell this time because this protein is intracellular.
My another question for permeabilization , with saponin or triton X100. I have never used it so no experience. I was looking for some who has done it.

'hope it helped.