Protein A chromatography

TPitarresi tpitarresi at aol.com
Tue Dec 12 20:37:16 EST 2000


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
time.

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.

regards,
Tina
Amersham Pharmacia Biotech






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