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).