Nov 24, 2022

Public workspaceLuciferase Activity Assay

DOI
dx.doi.org/10.17504/protocols.io.dm6gpj37dgzp/v1
  • 1Department of Health Sciences, University of Milan, Milan, Italy
Electra Brunialti
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpj37dgzp/v1
Protocol Citation: Electra Brunialti, Alessandro Maria Villa, Paolo Ciana 2022. Luciferase Activity Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpj37dgzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: November 22, 2022
Last Modified: November 24, 2022
Protocol Integer ID: 73113
Funders Acknowledgement:
The work was supported by the EU Joint Programme - Neurodegenerative Disease Research (JPND) project GBA-PaCTS, 01ED2005B
Abstract
Luciferase activity assay on cell extracts using Veritas (Turner) luminometer.
Lysate the cell using 1X Luciferase Cell Culture LysisReagent (Cat. E1531, Promega) following manufacturer instruction;
prepare Reaction Buffer (100µl for each sample): 500 µM luciferin (Cat E160E, Promega), 20 mM Tricine, 0.1 mM EDTA, 1.07 mM (MgCO3)4·Mg(OH)2, 2.67 mM MgSO4 in H2O, pH 7.8, with 33.3 mM DTT and 530 mM ATP.
put 20 µl/well of cell lysate into the white plate (Cat 3912, Corning);
turn on the luminometer (Veritas, Turner);
let the samples' plate and Reaction Buffer equilibrate at room temperature; Insert the plate into the instrument and prime the tube with the Reaction Buffer;
run the protocol on the luminometer (injection volume 100µl, delay before measurement 0s, integration time 10s)