Protein A chromatography
brucec at brucec.mv.com
Wed Dec 13 03:33:13 EST 2000
Thanks for this eminently helpful post!
> Hi Bruce,
> Try 2-4 M urea at neutral pH (filter with a .2 um filter, you don't want your
> cleaning solution to contain particulates) . You can try 2 M Guanidine HCl as
> well. If possible reverse the flow on the cartridge. 5 bed volumes usually
> does the job. Urea targets ppt proteins, it should have little effect on the
> binding activity of Protein A. Watch your flow rates and pressure. Urea will
> generate more pressure than most aqueous buffers.
> If the perseptive cartidge can handle alcohol, you can also try 20%-70%
> ethanol. Don't jump straight to 70%, you run the risk of forming bubles in the
> cartridge due to outgasing. Ethanol should target lipids and other hydrophobic
> contaminants. Again 5 bed volumes is usually enough. Like the urea ethanol is
> more viscous than most buffers, watch your flow rates and pressure. Protein A
> should be stable with an ethanol wash.
> You can also try a warm detergent wash. 1% detergent solution in water warmed
> to 37%. You will need extensive washing (15-20 bed volumes) to remove all the
> detergent. -- watch SDS it can reduce the binding capactity of Protein A over
> Try a very low pH wash-- Protein A is stable for short periods of time at pH 2.
> A You could use glycine HCL. Be careful-- Protein A will lose activity if
> left for an extend period. Also--watch out for the base matrix. You need to
> ask perseptive if it is stable to acid.
> Amersham Pharmacia Biotech
More information about the Proteins