Question: I was wondering if you could elaborate on why the Streptavidin-ZAP product recommends to be used at an equimolar ratio with the targeting reagent, when it is capable of binding up to four biotins/molecule?
Answer: It’s a question we get asked sometimes and it’s a good question.
You are correct that streptavidin is capable of binding up to 4 biotin molecules. However, when we created streptavidin-ZAP with the purpose of being a modular way of creating targeted toxins, we learned that the best general rule to follow was using a equimolar reaction. In theory, it is a 1:1 ratio of targeting molecule to streptavidin-ZAP, where we are most likely seeing an average of 1:1, but there is also the possibility of mixed ratios.
The amount of publications using the equimolar approach gave the desired results whether they were using a small biotinylated peptide or whole IgG. You’ll notice that depending on the MW of your biotinylated targeting agent, the amount of streptavidin-ZAP needed for the experiment can vary drastically and through in-house characterization, the equimolar approach still worked best.
Another reason we recommend a 1:1 ratio is based on our experience with our other secondary conjugates. It may be intuitive to think that using a higher dose of targeting agent would induce more cell death, but we found the opposite effect, where the excess, un-reacted targeting agent competed with the conjugated material for surface binding sites, which in turn decreased the amount of saporin being delivered. We have a publication (PMCID: PMC8952126 ) that also describes this observation.
Once you’ve created a baseline using the equimolar protocol and are more accustomed to how streptavidin-ZAP works in your application, please contact us if you feel more optimization is needed. It will be easier to help trouble-shoot when we are all working off the same protocol.
Related Products: Streptavidin-ZAP