Antibody Purification

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Mon Feb 21 11:06:13 EST 2005


Biplab Bose wrote:

> Hi 
> I am using MAbTrap Kit with HiTrap Protein G HP 1 ml column(Amersham),
> to purify a recombinant human antibody (IgG1). 
> 
> 1. Crude culture supernatant: ELISA O.D = 1.6/0.5 micro gm
> 2. Flow through: ELISA O.D = 0.3/0.5 micro gm

looks like you could use more gel for your sample or pass the sample
over the gel a second time as some of the antibody is left in the flow
through.

> 3. Eluted fraction: ELISA O.D = 0.6/0.5 micro gm

Either your antibody stays on the column or it is denatured during
elution.
> 
> As far as I understand the purified eluted fraction should give more
> ELISA reading than the original crude sample.

Correct, the purpose of protein purification is an increase in specific
activity (signal / protein amount).

> Can the neutralization buffer, used during purification, creat problem
> in ELISA?

Yes, you should do a buffer exchange into PBS before analysing. Dialyis
or gel filtration are suitable methods. Note that this should be done as
soon as possible after elution, to protect the antibody.

> How can I increase the yield of purification?

The solution depends on which of the above reasons cause the problem.
The buffer exchange you should do anyway. If that doesn't cure the
problem you could try and determine the yield of IgG (semi-quantitative
Western blotting or ELISA for total IgG). If the antibody stays bound to
the column, your yield will be low. If you can account for most of your
IgG, then there is a problem with denaturtion. Alternatively, try and
boil some of the eluted gel with SDS sample buffer and do a SDS-PAGE
with the supernatant: there should be little or no IgG.

Getting antibodies from a column is always a balance between harsh
condition that destroy activity and mild conditions that do not elute
them. The problem is particularly acute with monoclonal antibodies. One
possible solution is to use electroelution from the gel.



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