I need to prepare NBT and BCIP solutions to use in my Westerns (strep-tagged proteins detection using Strep-tactin AP conjugate). The company's (iba lifescience) protocol recommends to use 7.5% NTB in 70% DMF and 5% BCIP in DMF. Previously I used SigmaFast tablets and it worked well but now I want to make my own stock solutions.
I've ordered both chemicals from Sigma and preparation instructions are rather different: BCIP disodium salt is insoluble in DMF and only soluble in water at 50mg/ml. For NBT, instruction recommends to prepare 10 mg/ml stock which is only stable for 1-2 weeks. I suspect it is more stable in DMF but instruction says nothing about it.
Another thing is, the reaction buffer in iba protocol has pH 8.8 and in protocol from Sigma pH is 9.5. Does it have something to do with a solvent?
So, my questions are: can I dissolve BCIP in water and NBT in DMF and use them in one reaction? Does the pH of reaction buffer matter? What is the optimal proportion of BCIP : NBT?
I've found a lot of different protocols in the internet so I guess AP detection works in a broad range of conditions but I just want to make sure that I'm doing the right thing.