Plasmatic cell membrane isolation (URGENT!) - (Jan/18/2006 )
Good afternoon to you all!!!
I am working with tumour cell lines and I need to perform an assay in order to test if after adding a ligand, its receptor is internalized. So, I would like to isolate plasmatic cell membranes from all the rest of the cell (after cell lysis).
Could someone please send me a useful protocol for it? It is very urgent (I'm about to end my PhD and this is the last experiment!).
i have no experience with such an experiment, but do you realy think it's possible to dicern plasma membrane from organels' membrane?
i know that you can lyse your cells after fixation and recover membranes after ultracentrifugation in sucrose buffer at 100,000 g for 1h at 4C...
why don't you try confocal microscopy after permeabilisation?
Could you just look at a time course of surface expression of the receptor by flow cytometry or look at localisation by immunocytochemistry after adding the ligand? Or use a fluorescently-labelled ligand and use time-lapse microscopy to track internalisation?
All the best,
All the best,
your best shot would be to disrupt cells mechanically and then pellet them at low Gs to bring down the heavy fractions (plasma membrane/nuclei). Now this is extremely dirty since you'd be pulling down partially disrupted cells that have most of contents inside. You could theoretically try to isolate endosomes and control the purity of your fractions with endosome markes, but believe me you don't want to go that way.
If you have access to a fluorescence microscope and if you have a suitable antibody for immunocytochemistry you can incubate your cells with your primary antibody at room temperature (or even lower - 10C) so you block all the endocytic machinery. You can try 1:50 - 1:200 for 15-30min. This will mark all your surface receptors.
Then you incubate them back at 37C to allow endocytosis (you can do several timepoints and determine endocytic rates) and then after the desired timepoint you fix them with PFA. this will not permeabilize the membrane which is important.
Then you make a two step incubation with 2 secondary Abs, with 2 differnt chromophores attached. The initial staining will give you the receptors that remained at the surface.
Then you permeabilize the cells with detergent (Triton should be fine if your protein is not picky) and then incubate with your 2nd secondary Ab (with different color). The surface receptors will already be bound to your previous (first) 2ry Ab and only the internalized ones will be free and will bind this 2nd 2ry one.
Then you have a way to determine the ratio of internalized/surface or internalized/ total by making the fluorescence ratio of teh 2 different colors.
This may be the best way to start.
If you're not getting good results (don't forget to control for leakage during the incubation with the 1ry Ab and do all your staining controls) you can try biotynilation assays, where you biotinylate all surface proteins, let them internalize, block surface biotine and then access for internalized biotinylated receptors. this is more accurate because you access it by western blot and the analysis is more straightforward but the biochesmistry of it can give problems sometimes.