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.
1. Protein preparation
2. Nucleic acid preparation
3. In vitro biochemical assays
Table 1: Primers used to PCR amplify the gene fragments of Meiob and Mov10. The bold letters in forward and reverse primers are BamHI and NotI cutting sites; the italic bold letters in a reverse primer are XhoI cutting sites; the bold letters in boxes indicate the nucleotides corresponding to the point mutation R235A.
Fragments | Primers | Sequences (from 5’ to 3’) |
MEIOB-A | forward | CGGGATCCATGTTACTTTCTTTGATACACTTGCC |
reverse | ATGCGGCCGCTACTTTTAGCTGTTCCACTG | |
MEIOB-C | forward | CCGCGTGGATCCATGGAACCAAAATACTTTACAACTTCA |
reverse | CGATGCGGCCGCCTCTTTATTTTCTCTTATATAATTCAGTAG | |
MEIOB-E | forward | CCGCGTGGATCCATGTTACTTTCTTTGATACACTTGCC |
reverse | CGATGCGGCCGCTACTTTTAGCTGTTCCACTG | |
MEIOB-E-mut | forward | CTCTGATGTGGCAATAAATTTTAAC |
reverse | GTTAAAATTTATTGCCACATCAGAG | |
MOV10 | forward | GACGACGATGACAAGGGATCCATGCCTAGCAAGTTCAGCTGCC |
reverse | GCTTACTCAGCTAAGCTCGAGTCAGAGCTCATTTCTCCACTCTG |
Table 2: Sequences of DNA/RNA substrates used in this work.
For figures | Names | Sequences (From 5' to 3') |
Figure 2, 5 | 36 nt ssDNA | 5'-Biotin-GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT-3' (DNA) |
Figure 3 | 36 nt ssRNA | 5'-Biotin-GUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGUGU-3' (RNA) |
Figure 4 | 54 nt 5'-tailed dsRNA | 5'-Biotin-ACCGCUGCCGUCGCUCCG-3' (RNA) 5'-ACGAGGGAGACGAGGAGACGGAGCGACGGCAGCGGU-3'(RNA) |
The authors have nothing to disclose.
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 |