Hi all.
I'm using a biotinylated conotoxin to attempt to visualise voltage gated calcium channels. At the moment I'm getting such high background/non-specific staining that it's impossible to see anything.
Do I need to use some kind of endogenous biotin blocking agent? I'm also used to blocking non-specific staining during immunocytochemistry but do I need to block with something in order to prevent non-specific binding of my fluorescent streptavidin?
If I'm missing some kind of vital blocking step then any knowledge would be gratefully received!
Thanks,
Eve
Rockland has some pretty good tips on this, but be aware you'll still need to optimize wash times, buffer composition, etc for your prep.
https://rockland-inc.com/streptavidin-biotin-tips.aspx
Hi Eve,
I use this kit to block endogenous biotin:
https://www.thermofisher.com/order/catalog/product/E21390
That being said, I also perform normal blocking steps with serum as the same species of the secondary antibodies and 1% BSA. The fluorescent streptavidin should be highly specific to your primary biotin labeled antibody, but be sure to use a secondary controls to be sure you aren't seeing artifact from nonspecific staining. Are you using a detergent to permeabilize and wash? For ICC I use .1% Saponin and for tissues .1-2% TritonX. I hope this helps!
-Elliot
Vector makes a great Avidin/Biotin Blocking Kit ( Cat. No: SP-2001 ). This should really help cut down on your background. Other suggestions that might help are reducing the concentration of your secondary or reducing the concentration of your primary. Without the whole staining protocol, it is difficult to know for sure where the issue lies.
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