Biotin-streptavidin signal amplification - (Jun/02/2014 )
I'm using a biotinylated primary antibody, followed by PE-conjugated streptavidin. I've heard that using streptavidin and biotin can help to amplify signals, but I am right that this is only the case when the biotin is attached to the label? This seems to make sense as one streptavidin can bind four biotin molecules, but I've read conflicting information so I'm not too sure...
The available formats for biotin-streptavidin interactions are generally unidirectional. Biotinylated primaries, with avidinylated conjugates.
At the scales of sensitivity we're talking about, particularly with the most recent generation of digital cytometers, the kind of multivalent amplification you're talking about is more likely to lead to artifactual noise signal than real amplification.
Instead, try titering up the primary you're using, or shopping around for higher affinity mAb clones, if you're really concerned with sensitivity.
The signal amplification would come about because every molecule of biotin on the primary antibody may bind a separate molecule of streptavidin, which then brings with it one or more PE. If there are four biotin on the primary and all four bind the same streptavidin molecule (which has four biotin binding sites), then the signal amplification would be entirely at the streptavidin-PE level.
To recap, if the primary has <n> biotins, then it can bind up to <n> streptavidin molecules. If streptavidin is conjugated to <m> PE molecules, then one would expect a signal amplification that can scale up to <n> * <m>. In reality, the amplification is likely less due to steric hindrance. FYI: streptavidin ~ 60 kDa, PE ~ 250 kDa, antibody ~ 150 kDa.