Antibody purification

D.K. dk at no.email.thankstospam.net
Thu Aug 8 23:48:55 EST 2002


"Vayuputra" <vayuputra369 at hotmail.com> wrote:
>D.K.
>
>Thanks a ton.  That was great detail.  I'll try the protocol.  Any thoughts
>on possible hindrances of reduced glutathione (in eluted protein) while
>binding to Affigel?  I read somewhere that the chemistry of protein binding
>might be inefficient due to the presence of reduced glutathione.  I'm sure
>you overcame that problem, if it is real.

Glutathione is a tripeptide. Thus, it will naturally interfere with every 
protein immobilization chemistry except the ones that employ linkage 
through sugar modifications. With 20 mM in elution being typical, NH2
concentration is going to exceed activated group concentration 
(10 umol/ml=10 mM is typical). Yes, a thorough, thorough dialysis 
against a strong buffer (you get acid as reaction product) that 
does not contain amines is essential. 

DK



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