Removing annoying heavy chain detection in IP/blots

D.K. dk at no.email.thankstospam.net
Wed Aug 21 10:02:57 EST 2002


In article <3D637777.1BB122A0 at Fackelmayer.de>, Frank at Fackelmayer.de wrote:
>
>
>fred wrote:
>> 
>> When we do IPs and boil the beads in SDS to run a gel, we then probe the
>> blot for the protein/antigen of interest.  However, most of you should know
>> that the IgG heavy (55 kDa) and light (25 kDa) chains often interfere with
>> detection of proteins in this MW region.  Hence people often cross-link
>> their antibodies to Protein-A beads first.  We tried that, but still, a very
>> small amount of IgG got thru somehow.  Would people care to comment on:
>> 
>> - How to remove trace IgG from beads after cross-linking the antibodies to
>> Protein A, but before using them for the IP.  We don't want to do harsher
>> crosslinking as it might wreck the IgG binding to our target.  (We used DMP
>> (dimethylpimelimidate) to cross-link).
>> 
>
>DMP is fine.
>wash thoroughly after crosslinking, using stringent buffers to get rid
>of bound but not crosslinked IgG. 
>We use short washing steps (in a small disposable column from Biorad)
>with 200mM Glycine/200mM NaCl/made pH2.0 with HCl. This breaks the
>IgG/ProteinA binding when it is not covalent (ie crosslinked). It is
>essential to make these washing steps fast, and immediately neutralize
>the material afterwards, as longer acid treatment will ruin much of the
>antibody. 

Or you could use 3.5 M MgCl2 which removes unbound material as 
affectively but does not affect antibodies in a time frame of hours. 

DK





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