Primary cell culture: labelling in vivo - How to label a protein for Fluorescence Microscopy? (Dec/19/2005 )

Hi All,
I hope that anyone of you could indicate me whether and how is possible to label a single protein (such as a proteasomic subunit) in "living" cells and track the label by fluor. microscopy.
I would like to perform a time-laps experiment and follow the effect of the treatment on the re-comportamentalization of that target.

Thanks a lot in advance.

Davide
Temple Univ.
Philadelphia

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If it is a protein you can add endogenously one can label the protein with a kit. The only other way is to add the antibody to a living cell which complicates things. Otherwise you can make a plasmid with a floursent tag (such as YFP) and transfect it in (we use amaxa for primary cells). Hope this helps.

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Thanks a lot for your help.
I would to follow the migration of one of the subunit of Proteasome in a primary neuronal culture under a drug-treatment.
I need to keep a single cell under analysis during a time-lapse with a quite fluorescent microscope.
I am wondering if there is some methods that allows to check that tracking without interferring (or minimiz. the interference) of the antibody or -tag or whatever I have to use for this purpose.

I will appreciate any helpful suggestion you would give me.

Davide

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