purification of fc-fusion protein

D.K. dk at no.email.thankstospam.net
Wed Mar 6 09:30:54 EST 2002


mautino at dba.unito.it (beatrice) wrote:
>Hi all,
>I'm trying to purify a Fc-fusion protein using protein-G beads (from
>Santa Cruz) without good results.
>I directly add the beads to the conditioned media (diluted 1:4 in
>water), incubate for an hour at 4°C rotating, 

How much of the sorbent do you add? If the total volume is
much larger than ~10X volume of sorbent, the binding might not
be efficient enough.

>wash twice with PBS and
>then elute with 0,1M Na-citrate pH 3.5.

In my experience, 3.5 is not low enough. You have to go 
to 2.5-2.8 to get efficient elution. So, perhaps you do 
have binding? 

>Well, the beads are unable to bind the fusion prot, Why?
>Have you got some suggestions?

In any case I'd suggest doing it all in column format
rather than batch. 

DK




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