Date: Thu May 24 2007 - 03:48:23 EDT
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 <http://www.ablynx.com/> antibodies Guy Hermans, PhD Senior Scientist Ablynx NV Technologiepark 4 B-9052 Zwijnaarde Belgium guy.hermans@ablynx.com tel: fax: mobile: +32 (0)9 261 06 57 +32 (0)9 261 06 27 +32 (0)486 788 551 <https://www.plaxo.com/add_me?u=30065269879&v0=994426&k0=2009290972> Add me to your address book... <http://www.plaxo.com/signature> Want a signature like this? -----Original Message----- From: Theresa Liao [mailto:theresaliao@gmail.com] Sent: Wednesday, May 23, 2007 4:50 AM To: cyto-inbox 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 ----------------------------------------------------------------------- THIS E-MAIL MESSAGE IS INTENDED ONLY FOR THE USE OF THE INDIVIDUAL OR ENTITY TO WHICH IT IS ADDRESSED AND MAY CONTAIN INFORMATION THAT IS PRIVILEGED, CONFIDENTIAL AND EXEMPT FROM DISCLOSURE. If the reader of this E-mail message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify us immediately at ablynx@ablynx.com. Thank you for your co-operation. "NANOBODY" and "NANOCLONE" are registered trademarks of Ablynx N.V. -----------------------------------------------------------------------
