[Protein-analysis] Re: for purification

Dr Engelbert Buxbaum via proteins%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Wed Apr 25 15:43:34 EST 2007


Am 25.04.2007, 02:33 Uhr, schrieb jayaraman tharmalingam  
<ntr_jayaraman from yahoo.co.in>:

> Dear sir,
>                 i am try to purify the IgG fraction from rabbit serum. i  
> use the proteinA-CL agarose column for best fit to purify the IgG but i  
> encounter some problem with this column.
> 1) i unable to elute the IgG fraction by 0.1M glycine-Hcl buffer but i  
> try other option with 3M potassium isothiocynate, it elute some  
> reasonable concentration of IgG.
> 2) In ELISA (Antibody capture), i use 3M potassium isothiocynate elute  
> IgG but it not work well.
> so please somebody help me to resolve my problem.

Binding between IgG and Protein A is quite tight, elution requires harsh  
conditions which not all antibodies survive functionally intact.

You could try either elution whith some other mildly caotropic agent  
(iodosalicylate, MgCl2), or, more promissing, electroelution.

Another question: How did you purify the antigen for injection into the  
rabbit? If the antigen was denatured (e.g. after SDS-PAGE) the antibody  
may recognise only the denatured, but not the native antigen. Then a  
Western blot would work, but ELISA or immunoprecipitation would not.


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