Helicase Activity Measurement of a Target Protein Using Biotin-Labeled RNA Duplexes

Published: June 29, 2023

Abstract

Source: Yu, L. et al., In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions. J. Vis. Exp. (2019)

In this video, we demonstrate the procedure to determine the helicase activity of a target protein to unwind the biotin-labeled dsRNA substrate. The activity of the enzyme was identified by analyzing the electrophoretic mobility shift, followed by a chemiluminescence assay using chemiluminescent enzyme-conjugated streptavidin.

Protocol

1. Protein preparation MOV10 expression constructs Generate cDNA expression constructs encoding mouse MOV10. Set up the polymerase chain reaction (PCR) reactions for each fragment. Mix 1 μL of mouse cDNA (from C57BL/6 mouse testis), 1 μL of dNTP, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of DNA polymerase, 25 μL of 2x PCR buffer, and 18 μL of double distilled H2O (ddH2O) in a fi…

Divulgazioni

The authors have nothing to disclose.

Materials

Equipment
Centrifuge Eppendorf, Germany 5242R
Chemiluminescent Imaging System Tanon, China 5200
Digital sonifer Branson, USA BBV12081048A 450 Watts; 50/60 HZ
Semi-dry electrophoretic blotter Hoefer, USA TE77XP
Tube Revolver  Crystal, USA 3406051
UV-light cross-linker UVP, USA CL-1000
Materials
Nylon membrane Thermo Scientific, USA TG263940A
TC-treated Culture Dish Corning, USA 430167 100 mm 
TC-treated Culture Dish Corning, USA 430597 150 mm 
Microtubes tubes AXYGEN, USA MCT-150-C 1.5 mL 
Tubes Corning, USA 430791 15 mL
Reagents 
Anti-FLAG M2 magnetic beads Sigma, USA M8823
ATP Thermo Scientific, USA 591136
BCIP/NBT Alkaline Phosphatase Color Development Kit Beyotime, China C3206
CelLyticTM M Cell Lysis Reagent  Sigma, USA 107M4071V
ClonExpress II one step cloning kit  Vazyme, China C112
Chemiluminescent Nucleic Acid Detection Kit Thermo Scientific, USA T1269950
dNTP Sigma-Aldrich, USA DNTP100-1KT
DMEM Gibco, USA 10569044
EDTA Invitrogen, USA AM9260G 0.5 M
EDTA-free protease inhibitor cocktail Roche, USA 4693132001
EndoFree Maxi Plasmid Kit Vazyme, China DC202
FastPure Gel DNA Extraction Mini Kit Vazyme, China DC301-01
FBS Gibco, USA 10437028
FLAG peptide Sigma, USA F4799
Glycerol Sigma, USA SHBK3676
HEPES buffer Sigma, USA SLBZ2837 1 M 
KOAc Sangon Biotech, China 127-08-02
MgCl2 Invitrogen, USA AM9530G 1 M
NaCl Invitrogen, USA AM9759 5M
NP-40 Amresco, USA M158-500ML
Opti-MEM medium Gibco, USA 31985062
PBS Gibco, USA 10010023 pH 7.4
RNase Inhibitor Promega, USA N251B
Streptavidin alkaline phosphatase Promega, USA V5591
TBE Invitrogen, USA 15581044
Tris-HCI Buffer  Invitrogen, USA 15567027 1 M, pH 7.4
Tris-HCI Buffer  Invitrogen, USA 15568025 1 M, pH 8.0
Tween-20 Sangon Biotech, China A600560

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Citazione di questo articolo
Helicase Activity Measurement of a Target Protein Using Biotin-Labeled RNA Duplexes. J. Vis. Exp. (Pending Publication), e21437, doi: (2023).

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