Removing serum proteins bound to Plasma membrane? - (Dec/13/2006 )
Hi!
Can anyone suggest something to remove non-specific binding of serum proteins to the cells. I incubate my cells overninght in DMEM containing serum and next day wash it thoroughly with PBS. However, I still find many proteins (from the serum) after I wash my cells with Hepes buffer and then precipitate it with TCA.
Hi Paras,
Rather than trying to remove all serum proteins from the cell membrane, could you not include a control which will have similar treatment except your test condition?
Rather than trying to remove all serum proteins from the cell membrane, could you not include a control which will have similar treatment except your test condition?
I want to see a differential pattern in my control and test....But i get too many proteins in the control and then its hard to deifferentiate the ones present only in the test.....?? Do you think some buffer e.g. NaCl or KCl can do this job by just giving a wash with the buffer??
You said you tried PBS and it did not help; I don't think then any buffer will be able to do the job.
You might want to use serum free medium.
Thanks for your reply...But how many hours maximum can we keep the cells in serum-free medium.....??
I was just wondering if a high concentration of salt can remove some of the proteins non-specifically bound to the plasma membrane!!!
When I change from serum(+) to serum free media, I wash the cells with PBS and then add serum free media, wait 2-3 hr and then change to serum free media again. I know the cells take up the serum proteins and then secrete them back into the media, so if you don't change twice there will still be serum proteins present.
However, I am collecting the media for western blot, but this could probably also reduce the serum proteins bound to the cell surface for your purpose.
Hi! Zona Pellucida,
Thank you for your reply. I want to do a SDS gel and then see the differential pattern. Can you tell me what do you mean by serum-free media. I think its not the Invitrogen serum-free media!!
And how many hours you keep the serum-free media on the cells for the second time?? Did you ever try to run a gel and see if the serum proteins are not present now after two incubations with serum-free media??
Hi Paras,
Serum free medium which we are talking about here is a medium which doesn't contain serum but does contain alternatives to serum. If you google it, you will find many hits.
Hope this helps.
Thanks I find it with Invitrogen!!
Hi Paras,
I am pleased to know that.