Phage display against protein A immobilized target

Deshpande, Raj rajendra at amgen.com
Tue Mar 17 15:06:09 EST 1998


Ralf,

If the affinity of the tag for its antibody is good (low to high nM), it
is safe to say that you could pull down phage particles bound to the
target by antibody beads.  You may want to limit the 'bead selection'
step to a shorter period of time (e.g. 30-45 min. at RT) to avoid
non-specific phage binding. You may also prefer using magnetic beads to
agarose beads, since the pore-size of agarose may cause non-specific
trapping of phages.  I hope this helps the first steps of your
experiments.  Good luck.

-Rajendra


**************************
Rajendra V. Deshpande, Ph.D.
Mammalian Cell Molecular Biology
Amgen, Inc.
One  Amgen Center Drive
Thousand Oaks, CA 91320-1789
Tel. (805) 447-7821
Fax (805) 499-7464
E-mail : rajendra at amgen.com
**************************

> ----------
> From: 	Ralf Landgraf
> Reply To: 	landgraf at mbi.ucla.edu
> Sent: 	Tuesday, March 17, 1998 12:00 PM
> To: 	methods at net.bio.net
> Subject: 	Phage display against protein A immobilized target
> 
> Does anyone have experience with phage display selection against a
> target which has been immobilized by a protein A agarose bound
> antibody.
> 
> The protein I am selecting against has a C-terminal affinity tag for
> an
> antibody.  I would like to use this anchor as a means of attachment
> since I know that it will not interfere with the binding of protein I
> am
> selecting.  It also seems to me that I should get more selective
> binding
> in suspension compared to antibody  bound to microtiter plates.
> However,
> I am not so sure about the possible unspecific selection that might
> result from using agarose bound protein A as my matrix.
> 
> Does anybody have experience with this setup?  What is your favorite
> blocking reagent etc?
> 
> Thank you very much in advance for any input.
> 
> 
> 
> Ralf Landgraf
> Molecular Biology Institute
> UC  Los  Angeles
> 



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