Feb 02, 2025
RIGI ATPase Assay
- 1Washington University
Protocol Citation: Carolina Lopez 2025. RIGI ATPase Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9zybl3p/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
We use this protocol and it's working
Created: January 14, 2025
Last Modified: February 02, 2025
Protocol Integer ID: 118312
Abstract
Protocol for in vitro testing of RIG-I ATPase activity
Materials
For 10x Buffer:
1 M HEPES (Thermofisher, 15630080)
5 M NaCl (Thermofisher, A57006)
50 mM MgCl2 (Sigma Aldrich, M8787)
Nuclease Free H2O
RIG-I (Abcam, ab132502)
White 96 well plate (Millipore Sigma, MSSWNFX40)
0.6 mL PCR tube
Buffers and Protocol
Buffers and Protocol
1h 15m
1h 15m
Make 10x ATPase Sample Buffer:
HEPES 100 millimolar (mM)
NaCl 1.5 Molarity (M)
MgCl2 10 millimolar (mM)
For making 1 mL 10x ATPase sample buffer, mix:
100 µL of 1 Molarity (M) HEPES
300 µL of 5 Molarity (M) NaCl
200 µL of 50 millimolar (mM) MgCl2
400 µL of H2O
Mix components in a 0.6 mL PCR tube:
6 µL of nuclease Free H20
1 µL of 10 millimolar (mM) ATP
1 µL of 10x ATPase Assay Buffer
1 µL of IVT RNA 1 µg/µL
1 µL of RIG-I
As controls, replace either the IVT RNA or RIG-I with H2O.
Incubate the reaction at 37 °C for 01:00:00 in a thermocycler.
1h
Remove reaction from thermocycler and add 40 µL of H2O. Then transfer 50 µL of mixture to a well of a white 96-well plate.
Add 50 µL of Cell-Titer Glo 2 mixture and incubate at Room temperature for 00:15:00
15m
Measure luciferase activity within each well with 2 second gain.