Cellular localisation of a protein - (Feb/20/2012 )

Hi, just a quick question from me

I have to come up with some experiments to study a theoretical protein expressed in liver cells. We were first asked to determine whether the protein is expressed on the cell surface (I chose to use flow cytometry with the appropriate monoclonal antibody to the protein that is conjugated to a fluorophore). The next two questions are a bit strange though.

We are asked how we would determine 1) whether it is also present in any intracellular compartments; 2) identify those compartments. Both these questions carry equal weight. My instinct was to use immunocytochemistry to visualise the protein localisation within the cell, but this seems to be the answer to question 2. Is there any way I could determine whether the protein is present intracellularly, without identifying the compartments immediately? I was thinking of fractionating centrifugation, but it seems to be nearly impossible to simply get rid of the plasma membrane and preserve everything else.

Any help would be greatly appreciated

Subcellular frationation is a commonly used process - it does involve quite a few steps and tends to be targetted towards isolating particular compartments rather than isolating all compartments with equal success.

You are correct that ICC would work for q1, it would also work for q2 - you can do staining for particular compartments, e.g. DAPI stains DNA so you can identify the nucleus, cytochrome C stains the mitochondria, etc. However, you can also identify most of the subcellular compartments be normal brightfield microscopy if need be, it just takes some practice.