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

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

1. MOV10 expression constructs
   1. Generate cDNA expression constructs encoding mouse MOV10.
      1. 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 final volume of 50 μL.
         NOTE: The primers for the amplification of Mov10 gene fragments are listed in&#....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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