[Babcock_Hall]

I have been writing up a document on enzymatic stop-time (endpoint) assays for the benefit of students who are undertaking them for the first time.  In stop-time assays one lets the reaction run for a fixed time, then adds a stopping solution (or does something else to quench the reaction).  Often the assay is a spectrophotometric one using a chromogenic substrate and a product that is a chromophore.  One thing that I noticed from reviewing various textbooks and articles is that some authors recommend two kinds of blanks.  One is a no-enzyme blank, and the other is a zero-time blank.  My question concerns the rationale behind the zero-time blank, which contains all reagents but is stopped as soon as all of the reagents have been added.

The no-enzyme blank will be nonzero under at least two conditions that I can think of, when there is a contaminating chromophore in the substrate, and when there is a non enzymatic rate of reaction.  The zero-time blank will also be positive in the first case, but it will obviously not allow one to see whether or not the second case is a significant problem.  One author says that this control checks for interferences from the enzyme itself.  Any thoughts on the advantages of performing both controls, as opposed to just the no-enzyme blank?

[Yggdrasil]

Looking at the zero time blank vs the no enzyme control can be useful to checking whether your quenching step actually stops the reaction.  It's probably worth doing at least once to validate a particular assay, but once you have a result either the zero-time or no enzyme should suffice for a negative control.

[Babcock_Hall]

Yggdrasil,

Thank you; I hadn't thought about the issue of stopping methods.