1
$\begingroup$

If an accurate measurement of enzymologic quantities is needed, then following established methods in the field is necessary.

However, it is sometime of great usefulness to ballpark a value before committing to taking the necessary time for protein purification, or buying the necessary equipment/reagents. For such purpose a little "hack" can go a long way. This often involve using an available equipment to substitute for another (e.g. maybe I have a FACS readily availably, however no HPLC).

I am seeking examples of "unconventional" use of lab technique that can be used to determine enzymologic quantities ($K_d$, $V_{max}$, $K_i$ ...) as a first estimate -- whether you have partaked in such heresies, or have merely seen them used, possibly even in a non-formal/DIY environment.

$\endgroup$
1
  • $\begingroup$ With bacteria, we often use selective or differential media to determine presence or absence of certain enzymatic reactions. Would that sort of think be useful? Probably wouldn't be able to estimate an enzyme kinetics that way though. $\endgroup$ – MikeyC Sep 15 '20 at 14:32
2
$\begingroup$

If there is a way to:

Then you can get good mileage out of some serial dilutions and a OD600 meter. Even better with a plate reader.

Exact protocol will depend on the quantity under consideration, but mostly will amount to substituting around in the enzyme kinetics equations until the quantity of interest is a function of the concentration, and devise a scheme to cancel out the other unknown quantities (often substrate concentration ramp + starting OD ramp + curve fitting)

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.