Polyclonal Ab Purification

Dayanidhi Raman maraman at ksu.ksu.edu
Sun Apr 16 14:31:54 EST 1995


gt7680b at prism.gatech.edu (Paul Kenneth Farmer) writes:

>Dear Netters:

>     I have tried affinity purifying polyclonal antibodies using Affi-Gel
>10, but unfortunately the elution using acidic, basic, or chaotropic
>conditions failed to remove most of the bound antibodies or at least had
>inactivated them. I am considering three approaches:  (1) Inverting the
>column and eluting in the opposite direction to prevent antibodies from
>dissociating and then reassociating with the bound ligand; (2) Using a
>batch method instead of column purification; and (3) Using electroelution
>of bound antibodies off of the column. My best success to date has been
>the straight acid elution using a glycine-HCl buffer, pH 2.5. However, the
>activity and specificity of the eluted antibodies were still unacceptable.

>     Does anyone have any suggestions on how to improve this procedure? In
>particular, has anyone tried electroelution coupled with inverting the
>column so as to place the immobilized antibodies closer to the anode?

>     Any suggestions would be greatly appreciated!!!!

>     My E-Mail address is: gt7680b at prism.gatech.edu
Hi, collect your fractions in 1M phosphate buffer pH 8.0 when you elute your antibodies in Glycine, pH 2.3. Add azide and store as such at 4 C. Good luck.


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