4 replies to this topic

[Kami23]

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

[dan_]

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

[bob1]

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

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

[Kami23]

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

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

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

Thanks guys!

Yeah we have done the western for both tissue and cell line.  Now we need to know where the protein is in just the cell line.  I think I get what your saying Bob1 because I have had to do this with tissue samples too.  Is it basically the same principle just with fixed cells?  previosly I have just probed the tissue with antibody.

[bob1]

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.