Donald J. Fauss wrote:
>> I need help on concentrating secreted proteins from cultured primary cells.
> I've tried 70% EtOH precipitation but that didn't seem to work. I'll try
> AmSO4 precipitation next but I'm wondering if I should include say 0.1%
> TX-100 to help with the more hydrophobic proteins (maybe just to keep them
> from sticking to the wall of the centrifuge tube?). I'm collecting them in
> plain DME so I know the high salt concentration might prove a problem.
> Probably I could detect the proteins with silver staining .
It is not quite apparent what you wish to do with the protein, and that
is important for the choice of concentration method. If it is only small
samples for protein determination and SDS-PAGE, then the
Chloroform/Methanol precipitation method of Wessel & Fluegge is probaby
your best bet, alternatively use the Phenol/Ether extraction method of
Fauve et al. In the old days Trichloroacetic acid (sometimes with
Desoxycholate as carrier) was used, but this method is now largely
superseeded by those mentioned above. Ammonium sulfat would be the wrong
choice for all electrophoretic methods (the increase in conductivety can
make your gels literaly boil).
If however you are dealing with large volumes for preparative work, and
if you need functional, non-denatured protein, then Ammonium sulfate
(usually carried out without detergent, by the way) or Aceton
precipitation may be a good option. Dialysis against Polyethylene glycol
8000 or ultrafiltration in an stirred cell under Nitrogen pressure
(Amicon has such devices) are other options. And of course you could try
any of the concentrating chromatography steps (ion exchange, affinity,
hydrophobic interaction, Hydroxyapathite ect), which would serve in
purification at the same time.