Localisation study - (Jun/22/2009 )

Hey all,

Right I have this problem but have no idea where to start... and I have to do it before I can start my RNAi work.

I have my cells growing in culture at the moment so they are on their way I need to know where my protein lies in the cell but (and here is the big problem) they are suspension cells. I understand how I could probe for my protein with monolayer cells but what would I do to use the suspension cells? Someone mentioned special coverslips or something but they left before I got a chance to pick their brains :S

Hi,

Have you ever though to make nuclear/cytoplasm/membrane/cytoskeleton fractions of total proteins that come from you cells? After this, simply make a western to see where your protein is located.

Qiagen have a kit that work well for this application. Qproteome Cell compartment kit.

Kami23 on Jun 22 2009, 03:16 AM said:

Hey all,

Right I have this problem but have no idea where to start... and I have to do it before I can start my RNAi work.

I have my cells growing in culture at the moment so they are on their way I need to know where my protein lies in the cell but (and here is the big problem) they are suspension cells. I understand how I could probe for my protein with monolayer cells but what would I do to use the suspension cells? Someone mentioned special coverslips or something but they left before I got a chance to pick their brains :S

Do a cell clot and fix it, section, stain etc.

Make a smear of your cells on a slide, fix and ICC.

bob1 on Jun 23 2009, 02:02 AM said:

Yes, just the same as IHC, but on cells instead of tissue. It is important not to fix (2% PFA for 15 min should work) for too long for this though, cells aren't like a lump of tissue.