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 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…

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