Protein G sepharose vs prot A with bridge

Toumy Guettouche tguettou at newssun
Thu Jan 23 15:14:08 EST 1997



On Thu, 23 Jan 1997, Mike Holloway wrote:

> Does anyone out there have experience enough to suggest which is better?  I
> want to immunoprecipitate with a monoclonal antibody.  It looks as though
> protean G sepharose should be the choice for substrate, but I note that most
> protocols I've seen use a rabbit anti-mouse bridge and protean A sepharose.
> Is this better than prot G?
> 
> Thanks,
> Mike Holloway
> holloway at cris.com
> 
> 
The difference of Protein A vs Protein G is the binding specificity. 
Antibodies from certain species bind only weakly or not at all to Protein 
A but bind strongly to Protein G and vice versa. Sometimes they have 
equal binding specificity and then it depends on the pH you are using, 
since they have different pH optima for binding. I found the 
Manual/Catalog from Pierce quit useful since they discuss this topic 
extensively.  
I use Protein G regularly without any problems to bind  mouse 
monoclonal antibodies for subsequent IP. But beware, you have to check out 
the subclass(IgG1a...etc.) , because some do not bind to Protein G.
I also have used bridging antibodies, but besides more work, they also 
give you more nonspecific binding(depending on the quality of the ab). 
Therefore I usually try to stay away from this method.

Hope that helps.

Toumy




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