[Histonet] Re: Biotin conjugated Antibodies -- can we skip biotin??

[Gayle Callis]

Yes, you do not need more biotin in the system.  When you work with 
biotinylated primaries, all you need to do is use Strepavidin-HRP or 
Strepavidin-AP, followed by the appropriate chromogen.

You eliminate the secondary conjugated to biotin completely, and also the 
ABC complex, and but be sure to use a good reliable Strepavidin-HRP or 
AP.  Southern Biotechnology has these as does Biosource/TAGO.  The primary 
conjugated to biotin provides all the biotin you will need.

One of the perks of working with biotinylated primaries - it shortens the 
protocol!

IF you use Vectors ABC complex, DON"T or you will experience loss of 
sensitivity and have poor staining.  You will still need to do the 
appropriate blocking, i.e. avidin/biotion (OR Strepavidin/biotin block from 
Vector)  normal serum, proper diluents with serums, etc. to avoid 
background problems.

We do biotinylated primaries almost exclusively and with great success for 
murine CD markers IHC and IFA (using Molecular Probes Strepavidin-Alexa 
fluorophore conjugates.)  If you want to discuss this more, contact me 
privately.

At 01:23 PM 7/26/2005, you wrote:
>Hello,
>
>Just a general question to help increase my knowledge:
>
>If I had an antibody that was conjugated to biotin -- say for example, a 
>goat anti-mouse P-selectin (biotin) -- can I just take a rabbit anti-goat 
>conjugated to AP and run IHC that way -- hopefully avoiding the ABC step 
>and background OR does the biotin from the primary Ab block secondaries 
>from coming in?
>
>Thanks.

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)