[Histonet] direct-conjugates for IHC

[koellingr <@t> comcast.net]

Jennifer, 



In the past, upon occasion when need arose, I'd use directly conjugated primary antibodies.  Mainly biotin or dig or peroxidase but also FITC directly to primary.  Then can look at it fluorescently or come back with an anti-FITC of some kind (several are good). Used these for my flow but also a lot of IHC.  Have never tried the Texas Red route for IHC.  What you say is true; direct conjugation can end up giving weaker signal but it doesn't have to be so bad.  1) If you are doing the conjugation, that is great and you have a chance to play with the system.  Conjugation can result in near loss of antibody to bind or can result in good Ab binding or if you have the knack it can result in little loss of binding.  For flow, the conjugation is not critical since as long as you don't ruin the antibody, it will work since flow reagents are generally used in excess since there is no background (the background is plain sheath fluid) although certainly you can put in too much.  For IHC you need to be gentler in conjugation.  Don't know your company  but if a biotech you might have a BiaCore machine  (measure affinity of Ab's) or there are other ways to measure affinity.  So I would always keep an aliquot of native antibody and after I did my conjugation, give those people both samples to measure affinity (or how much loss).  Tell me immediately if I lost too much Ab binding ability because of my (hopefully not poor) conjugation.  In general yes, directly conjugating your primary can weaken signal but it doesn't have to be as much as conventional wisdom or lore makes it. 



Raymond Koelling 

PhenoPath Labs 

Seattle, WA 


----- Original Message ----- 
From: "Jennifer Anderson" <janderson <@t> halozyme.com> 
To: histonet <@t> lists.utsouthwestern.edu 
Sent: Monday, May 4, 2009 12:04:57 PM GMT -08:00 US/Canada Pacific 
Subject: [Histonet] direct-conjugates for IHC 

Hello everyone! 

  

I work with a scientist who insists on using primary antibodies directly 
conjugated with FITC or Texas Red for IHC.  In my past experience these 
directly conjugated antibodies didn't give a strong enough signal for 
use in IHC (I've seen them used only for FACS analysis).  I would 
appreciate your professional comments. 

Thanks again, so much, for your insight! 

  

Jennifer M. Anderson, Scientist 

Halozyme Therapeutics, Inc. 

11404 Sorrento Valley Road 

San Diego, CA 92121 

858-704-8333 

janderson <@t> halozyme.com <mailto:janderson <@t> halozyme.com> 

  




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