Fw: Can FACS Ab's sterically hinder each other?

David.C.McFarland@gsk.com David.C.McFarland at gsk.com
Fri Aug 24 17:01:32 EDT 2007

I can up the ante on weird Ab interactions.  I've seen instances where 
single stains work, dual stains don't and sequential staining only works 
if you go in the right order!

David McFarland
Principal Scientist
----- Forwarded by David C McFarland/PharmRD/GSK on 08/24/2007 04:57 PM 

"Preffer, Frederic I., Ph.D." <PREFFER at HELIX.MGH.HARVARD.EDU> 
23-Aug-2007 20:19
"Cytometry Mailing List" <cytometry at flowcyt.cyto.purdue.edu>

RE: Can FACS Ab's sterically hinder each other?

I think it is possible you will see inhibition. You can certainly see the 
of   staining either anti- TCR alpha-beta [or gamma-delta] vs CD3 epsilon. 
two TCR chains are ensconced within the CD3 chains and thus show steric
opposition. I do agree with Tom that trying the staining individually and
simultaneously will give you the answer


-----Original Message-----
From: Tom Knight [mailto:thknight at ucdavis.edu] 
Sent: Tuesday, August 21, 2007 4:39 PM
To: cyto-inbox
Subject: Re: Can FACS Ab's sterically hinder each other?


I seriously doubt there will be any detectable degree of steric 
hindrance.  Why don't you do the experiment and find out?  Stain one 
tube with the two antibodies, one with just Ab#1, and one with just 
Ab#2.  If you see a significantly reduced distribution of fluorescence 
in either channel on the double stained cells compared to the 
corresponding single stained tube, please let us all know about it! 

Make sure you are correctly (and identically) compensating all three 
samples before making the comparison.  If you do see that one of the two 
Abs has reduced binding in the combination tube, you could circumvent 
the problem by staining first with that Ab, and then add the other one 
after 20 minutes or so.  But I'm betting you don't need to -- unless 
possibly this is a special application where you are trying to measure 
small changes in the level of expression of your proteins, e.g. a factor 
of 2 or less.

  -- Tom
Pieter Bogaert wrote:
> A collegue in our department had a question concerning sterical 
> hindering of Ab's. I have never encountered such a problem, but I'm 
> not that experienced, so perhaps some of you have?
> Anyway, this is his question:
> I have a transmembrane protein that is expressed abundantly at the cell
> surface. The extracellular part of this protein is only 24 amino acid
> residues long. In FACS experiments a monoclonal antibody directed 
> the extracellular part of this protein work very well and specific. I
> would like to perform a double staining in FACS using this monoclonal ab
> together with a second antibody that is directed against an equally 
> (molar
> amount) abundant transmembrane protein but with an extracellular part 
> that
> is much larger: 550 amino acid residues. Is it possible or likely that 
> abs
> binding the large protein at the surface of the cell will sterically
> hinder the abs specific for the short extracellular part of the second
> protein? One ab is rabbit, the other mouse, both IgGs.
> Thanks on beforehand!

Tom Knight    530-205-6885 cell
		       916-734-8532 lab
		       916-734-4796 fax
Laboratory of Dr. David Asmuth
Infectious and Immunologic Diseases
University of California, Davis -- School of Medicine

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