Protein G sepharose vs prot A with bridge
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?
> 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
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.
More information about the Methods