On/Off rates of Rec. Fabs?

H. David Fischer davefischer at delphi.com
Wed Mar 8 21:46:17 EST 1995


Mxm <pemanuel at umabnet.ab.edu> writes:
 
>I have produced a set of recombinant Fabs using Phage display.  Now I was
>wondering if there was a way to screen them for On/Off rates.  Must I
>purify the Fabs from the bacterial supernatents in order to get this
>information?
>We are planning on subcloning many of the clones into a Histidine tagged
>vector for Ni+NTA column purification.  I would like to avoid having to
>subclone all of them since there are so many positives.  I would prefer to
 
Peter,
It's great to hear you have "so many positives" from the phage display system!
If you have access to either a Pharmacia BIAcore or a Fisons IAsys you can
generate those kinetic constants easily from your E.coli sups and very
possibly without needing to do much or any prior purification.  We have used
both systems with good success and usually get comparable values with each
for duplicate samples.  Because these machines measure binding in real time,
the rate constants fall right out.  With most other techniques count on
having to do multiple samples and multiple time points.  Also, the two
biosensor systems don't require any labels or affinity tags on your antibodies.
That will save you from the mess of labeling or subcloning into vector with
a fusion tag.  Finally, His tags are great for IMAC purification, but not so
hot as an affinity tag in many assay formats.  If you do have to go that
route, the commercial FLAG (Kodak) or E-tag (Pharmacia) systems have both
worked well for us and there are several others.  Just my 2 cents, anyway.
 
Dave Fischer, The Upjohn Company
hdfische at intnet.upj.com



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