Jump to main content
Jump to site search
PLANNED MAINTENANCE Close the message box

Scheduled maintenance work on Wednesday 21st October 2020 from 07:00 AM to 07:00 PM (BST).

During this time our website performance may be temporarily affected. We apologise for any inconvenience this might cause and thank you for your patience.


Issue 17, 2014
Previous Article Next Article

BRET-linked ATP assay with luciferase

Author affiliations

Abstract

Taking advantage of BRET, a mutant firefly luciferase with higher pH- and thermo-stability than the wild-type could be coupled with the red-emitting fluorescent protein of mCherry in both a fused and unfused format. The BRET pair allows >40% of the light emitted to be red shifted over 600 nm to the mCherry acceptor wavelength. Taking the expected quantum yield for mCherry (0.22), a good fit to predicted light transfer is shown, with no other losses. Two measurements are considered for ATP determination: (a) a ratiometric technique for ATP measurement using both donor and acceptor emission intensities, making the calibration slope independent of protein concentration in a broad range. This measurement was limited by the BRET efficiency and the low quantum yield of the mCherry acceptor, but this detection limit might be improved with other fluorescent proteins with higher quantum yield. The fused BRET pair also resulted in a small increase in the BRET ratio. (b) An ATP dependent shift in the wavelength maximum using just the acceptor mCherry emission was also proposed for ATP determination. This did not require a high BRET efficiency and only uses emission above 600 nm to obtain the acceptor emission maximum, but not its intensity; it is independent of protein concentration across a broad range. This offers a novel and robust method for determination of ATP between 10−11 to 10−5 M with an easy baseline calibration with ATP concentration >10−4 M.

Graphical abstract: BRET-linked ATP assay with luciferase

Back to tab navigation

Article information


Submitted
05 Mar 2014
Accepted
01 May 2014
First published
21 May 2014

Analyst, 2014,139, 4185-4192
Article type
Paper
Author version available

BRET-linked ATP assay with luciferase

G. Borghei and E. A. H. Hall, Analyst, 2014, 139, 4185
DOI: 10.1039/C4AN00436A

Social activity

Search articles by author

Spotlight

Advertisements