Format

Send to

Choose Destination
See comment in PubMed Commons below
Talanta. 2013 May 15;109:141-6. doi: 10.1016/j.talanta.2013.02.007. Epub 2013 Feb 8.

Use of hGluc/tdTomato pair for sensitive BRET sensing of protease with high solution media tolerance.

Author information

1
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

Abstract

Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (Δλ:~110 nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1 pM in pure buffer and 3.3 pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.

PMID:
23618151
DOI:
10.1016/j.talanta.2013.02.007
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center