Re: How to choose antibody for FACS

Hi Theresa,

Guy has explained very well the differences in antibody specificities.	We
have found no clear and consistent rule of thumb for which ones work in flow
if not previously tested.  The easiest way is to call the company that has
the antibody and ask for a sample with the promise you will tell them if it
works in flow.	I think the companies would be happy to send a test sample
as they might get your business and they would certainly get data without
having to test it themselves.

Randy T. Fischer, NIH/NIAMS
B Cell Biology Group
9000 Rockville Pike
Bldg 10, Room 6D50
Bethesda, MD 20892
(301) 594-3537 (voice)
(301) 402-2209 (fax)



From: Guy Hermans <Guy.Hermans@ablynx.com>
Date: Thu, 24 May 2007 09:48:23 +0200
To: cyto-inbox
Conversation: How to choose antibody for FACS
Subject: RE: How to choose antibody for FACS

Hi T.,
some antibodies, and I'm referring to monoclonals specifically, recognise
non-continuous "3D" epitopes on the surface of a protein, or even on two
adjacent non-covalently bound subchains. Denature and/or reduce the antigen
(as in SDS-PAGE/Wester Blot etc.) and they utterly fail to detect the
antigen(s) on the blot. Those could be good in FACS (if the epitope isn't
hidden by accessory chains proteins/inaccessibly close to membrane etc,),
but not in blots.
Some other mAbs will actually recognise linear epitopes buried in the core
of a protein, or recognise such linear sites on the surface of the protein
but which are normally interacting with other proteins. In denaturing
conditions run WB those will do just fine, but will not bind the
physiologically relevant state of the protein. Hence, that category of
monoclonals won't the useful for FACS but is for WB.
Therefore, FACS untested mAbs suitable for WB may or may not work for you
(and vice versa), for good reasons.
Incidentially, polyclonal antibody preps usually consist of such a diversity
of different fine specificities they will collectively bind both states of
the protein, even though different subpopulations of the polyclonal won't
bind one or the other.
I'll leave this lecture at that, for now ;-)
Guy
	    Next generation therapeutic       antibodies
<http://www.ablynx.com/>
      Guy Hermans, PhD
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>  
>  
> -----Original Message-----
> From: Theresa Liao  [mailto:theresaliao@gmail.com]
> Sent: Wednesday, May 23, 2007 4:50  AM
> To: Cytometry Mailing List
> Subject: How to choose  antibody for FACS
> 
> Hello everyone,
> 
> I have a question  regarding choosing a suitable antibody for FACS analysis.
> 
> Most of the  time, I can find antibodies that have been tested for FACS
> analysis by the  manufacturer or by some one else. However, sometimes I
> encounter an antibody  that I need which has not been tested for FACS. I am
> wondering what is the  difference between antibodies used in different
> applications such as ELISA,  RIA, IHC, and FACS? Are there antibodies suitable
> for one application but not  the other?  If I have to choose an antibody for
> FACS that has not been  tested specifically for FACS but has been tested for
> other applications, what  would be my best bet?
> 
> An example as below (this is what I think up in  my mind...)
> 
> Antibody one: tested for ELISA and RIA
> Antibody two:  tested for IHC (formalin fix parafilm blocked)
> Antibody three: tested for  immunoprecipitation
> 
> Which one would I choose if I need an antibody for  FACS? Or it is trial and
> error?
> 
> I appreciate your help.  Thanks!!
> 
> Theresa Liao
> 
> 
> -- 
> Yu Huan (Theresa)  Liao
> PhD Candidate, UBC Experimental Medicine Program
> Ike Barber Islet  Transplantation Laboratory, Vancouver General Hospital
> 
> "I think prime  numbers are like life. They are very logical but you could
> never work out the  rules, even if you spent all your time thinking about
> them"  Mark  Haddon, The Curious Incident of The Dog in The Night-Time


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