Hi Everyone,

I need to set up a protocol for TBO quantification of polyphosphate (PolyP) as a product reaction of thermostable Polyphosphate Kinase (TePPK). I set up a reaction with ADP, PolyP and TePPK, let it incubate for one hour at 60°C collecting samples every 15 minutes and putting them at -20°C to block TePPK activity. I’d like to understand how set up the quantification of the PolyP using Toluidine Blue O assay (I’m using Sodium polyphosphate -Graham’s salt- from Merck, which should have 25 phosphate residues); I found different guidelines about this analysis (metachromatic shift established by measuring the absorbance at 530 and 630nm, decrease in absorbance at 620nm by metachromatic reaction of TBO with poly P) but I’d like to have more detailed informations about:

- TBO solution preparation

- PolyP standard curve preparation

- How the reaction should occur (timing and temperature)

- How the reaction, in terms of PolyP quantification, should be measured

Hope I gave you clear explanation of my request! Thank you all! Alessandra