I'm not sure I entirely understand your problem, but it sounds like your
eluted purified antibody shows up only as a light chain band on PAGE.
What specifically are your elution conditions to get the Ab off the
affinity column?. You really might have heavy-light chain dissociation
during elution if your conditions are harsh or mildly reducing; despite
your finding that another antibody comes off intact doesn't mean that your
Ab of interest is going to act the same way (all MAB are different!). You
might wish to try an affinity purification using ProA or ProG agarose;
these would probably allow gentler elution conditions. Does your purified
product bind to its antigen? What is the specificity?
On 26 Aug 1993 BOTDJL at LURE.LATROBE.EDU.AU wrote:
> Date: 26 Aug 93 10:45:23 GMT
> From:BOTDJL at LURE.LATROBE.EDU.AU> To: immunology at net.bio.net> Subject: monoclonal oddity
>> Dear Immunologists,
>> I have a peculiar problem with regards to a monoclonal Ab. This Ab recognizes
> specifically my target protein (and no others) only when that protein is
> present in sufficient amounts ie detectable by coomassie blue staining. I have
> recently purified my monoclonal Ab from a spinner culture supernatant by
> passing the supernatant over an affinity column which has Ab bound to it that
> recognizes the kappa light chain of mouse monoclonals.
>> When I ran the eluted products on a PAG only light chains could be visualised
> on a coomassie blue stained gel. I know that the column is not stripping the
> light chains from the heavy chains becuase I passed ascites fluid of a
> different MAb over the same column and the heavy chains were easily detectable.
> This would mean therefore that I have seen specific binding of light chains
> only, to a target protein.
>> I am interested in all feedback that I may get with regards to this problem. I
> am not an immunologist, but one very frustrated Ph.D student.
> Darren Lowen.
>botdjl at lure.latrobe.edu.au>