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…
Declarações
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