precipitation of secreted protein from growth media - (Apr/12/2010 )
Hi,
I am growing some glioma cell lines and am interested to check if the protein of my interest is secreted into the media. for this I plan to grow the cells over a period of 3-4 days and then collect the media and precipitate out the proteins from it and run a western blot on it, as well as the lysate from the cells.
Is thsi the correct way to do it? Has anyone done it earlier? and which method should I use for precipitation of proteins from teh growth media, Aceton or TCA? is one better than the other?
-Anshul-
Anshul on Apr 12 2010, 04:02 PM said:
Hi,
I am growing some glioma cell lines and am interested to check if the protein of my interest is secreted into the media. for this I plan to grow the cells over a period of 3-4 days and then collect the media and precipitate out the proteins from it and run a western blot on it, as well as the lysate from the cells.
Is thsi the correct way to do it? Has anyone done it earlier? and which method should I use for precipitation of proteins from teh growth media, Aceton or TCA? is one better than the other?
I am growing some glioma cell lines and am interested to check if the protein of my interest is secreted into the media. for this I plan to grow the cells over a period of 3-4 days and then collect the media and precipitate out the proteins from it and run a western blot on it, as well as the lysate from the cells.
Is thsi the correct way to do it? Has anyone done it earlier? and which method should I use for precipitation of proteins from teh growth media, Aceton or TCA? is one better than the other?
Neither acetone nor TCA, but ammonium sulphate is the generally recommended technique. You do it by a number of rounds of dissolving solid Amm. SO4 into the solution and spinning down any precipitate formed. This precipitate will contain different fractions of the total protein content of the media. The beauty is that you can resuspend the pellet and proteins will typically still be native. The fractions can be run on a gel.
In order to keep the volumes manageable, it is often best to reduce the volume by tangential flow filtration or ultrafiltration; this also gets rid of salts and small molecules.
Good luck with it.
-swanny-