Antibody purification

[D.K.]

"Vayuputra" <vayuputra369 at hotmail.com> wrote:
>Can anyone suggest me the most efficient way to purify polyclonal antibodies
>from serum bleeds?
>
>We obtained polyclonal serum (from rabbits) against a His-tagged protein I
>expressed in E.coli.  I also have made a GST-fusion construct with a vague
>idea of overexpressing the GST-fusion protein and affinity purifying
>specific antibodies from the serum.  I'd like to do the affinity
>purification in the best (and hopefully the least cumbersome) way possible.
>Any and all help in this regard would be greatly appreciated.  Or, please
>let me know if you are aware of any alternatives.

Different people probably have very different opinions about what is the best and 
most efficient way of doing it. I've done it countless times and here is what I do.
Yes, if possible, use a different fusion protein from the one served as antigen -
contaminants will be different and thus Ab prep will be more specific. 

1. Covalently immobilize your protein (GST fusion in your case) on [usually]
agarose carrier. I tend to use Bio-Rad's Affi-Gel 10 or 15 or an equal parts
mix of 10/15 (Affi-Gel's capacity is lower than most activated sorbents 
from other manufacturers, and this usually results in cleaner preps). I am 
sure other coupling chemistries and other suppliers can give adequate 
results too. Couple ~ 3 mg protein/ml. Block with ethanolamine or, if it is not
available, glycine. 

2. Assuming you want IgG fraction free of IgM/IgE (almost always a good 
idea), buy some ProteinA-agarose/Sepharose.  I use Pharmacia stuff 
bought in smaller quantities from Sigma. 

3. Decide on your first purification step. Purity-wise it is better to start with 
the most specific step first. Thus, if you have an ample supply of antigen
affinity column, start with it. When antigen is hard to come buy (non-bacterial
expression or native protein), out of considerations of having antigen column
last longer, do total IgG purification first followed by an affinity purification. 

4. First step is either antigen affinity or protein A affinity. Home-made gravity 
columns packed in syringes or leftovers from Qiagen DNA preps work just 
fine. If antigen allows, room temp is OK. Adjust pH of serum to ~ 8.0 (for 
example, 1/10V of 1.0 M 10X Tris stock), if antigen allows, add NaCl to 
250 mM salt and/or ~ 0.5% detergent (like Tween 20). Standard run/wash to 
the point of < 10-30 ug/ml protein in the wash (omit detergent from washes 
beyond first several column volumes), elute step-wise with 3-5 column 
volumes  in one column volume steps with 5-15 min delay between. 
In either case (antigen or PrA columns) good eluents are 3.5 M MgCl2,
non-PHed or 100 mM glycine pH 2.5 with subsequent neutralization to 
pH >7.0 with 2.0 M Tris, pH 9.0 . Mg2+ elution is milder on Ab, pH elution
is more convenient (MgCl2 elution requires dialysis!). The choice is 
dictated by a sequence of columns used, antigen's properties and 
convenience. Whenever possible, I have this step as antigen column
eluted with MgCl2, followed by o/n dialysis into 125 mM Tris, 350 mM
NaCl, pH 8.0

5. Do a next column step. All the same considerations apply. If serum is 
good and everything is done right, in typical purification from 20 ml serum 
you get ~ 5 mg of very pure and specific IgG fraction at concentration 
1.5-3.0 mg/ml (rabbit IgG absorb 280 nm as ~1.4 OU at 1 mg/ml and 
react in Bradford test ~3.5X better than BSA). 

This is a two days, not too involved work, and I have had nothing but 
success with this kind of purification, the antibody being very specific 
and extremely inhibitory/potent in functional assays and staining strictly 
one band in Westerns of total cell lysates. 

DK