what cell line to use for visualizing membrane localized protein - (Oct/08/2008 )

Dear all,

I work with a cytosolic protein.
to this protein i attached a peptide that should induce plasma membrane localization.
i transfect this construct (peptide-bound) into HEK293 cells.
after staining with specific Ab i check for the localization to the plasma membrane using confocal microscope.

I can not clearly see that my protein localizes at the plasma membrane.

i would like to know whether HEK cells are ok for studying membrane localized proteins. If not what other cells should i use?
And is there any marker i can co stain to better see the plasma membrane?

Thank you in advance for support!

sarah

Pretty much any cell line should be OK for this. 293 are rather small though, you might be better off with a larger cell line like SaOS-2 or a fibroblastic line.

Before you go stressing about plasma membrane markers, make sure that your expression construct is working the way it should. Can you see any signal from the expressed protein? Is the protein expressed endogenously, if so, how much is expressed and will this interfere with what you want to see?

Molecular probes(Invitrogen) has a wide range of different types of markers available, they are typically very good, but i don't know specifically about plasma membrane markers.