Split Luciferase Complementation Assay to Identify Specific Protein-Protein Interactions
Published: April 30, 2023
Abstract
Source: Wiertelak, W. et.al., Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay. J. Vis. Exp. (2020)
This video demonstrates the split luciferase complementation assay to detect protein-protein interactions. In this assay, the proteins of interest are tagged to small and large fragments of the luciferase enzyme. When the proteins interact, the large and small fragments combine to form an active enzyme complex, which in the presence of a specific substrate, releases bright luminescence that can be measured.
Protocol
1. Transient transfection of the expression plasmids into the cells Harvest the adherent HEK293T cell culture by trypsinization and resuspend the cells in a dedicated complete growth medium. Plate the cells (2 x 104/100 µL/well) onto a clear bottom, white side 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls including replicates. NOTE: Attempt to use only the inner 60 wells of the plate to minimize thermal shifts …
Açıklamalar
The authors have nothing to disclose.
Materials
| 0.25% trypsin-EDTA solution | Cell culture | ||
| Adherent mammalian cell line | Cell culture | ||
| BioCoat Poly-D-Lysine 96-well White/Clear Flat Bottom TC-treated Microplate, with Lid | Corning | 356651 | Plastic ware |
| Cell culture centrifuge | Plastic ware | ||
| Cell culture supplements (heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptomycin) | Cell culture | ||
| CO2 incubator | Device | ||
| Expression plasmids encoding protein(s) of interest not tagged with NanoBiT fragments | Assay | ||
| FuGENE HD Transfection Reagent | Promega | E2311 | Assay |
| GloMax Discover Microplate Reader (or a different luminescence microplate reader) | Promega | GM3000 | Device |
| Growth medium dedicated to the cell line used | Cell culture | ||
| Materials and reagents for standard molecular cloning (bacteria, thermostable polymerase, restriction enzymes, DNA ligase, materials and reagents for nucleic acid purification) | Assay | ||
| NanoBiT MCS Starter System | Promega | N2014 | This kit contains vectors enabling tagging of the proteins of interest with NanoBiT fragments at different orientations as well as the control plasmid encoding HaloTag protein fused with SmBiT and a positive control plasmid pair. |
| Nano-Glo Live Cell Assay System | Promega | N2011 | This kit contains furimazine, which is a substrate enabling detection of the NanoLuc activity in living cells, and a dedicated dilution buffer. |
| Opti-MEM I Reduced Serum Medium, no phenol red | Gibco | 11058021 | Cell culture |
| Orbital shaker | Device |
Etiketler
Bu Makaleden Alıntı Yapın
Split Luciferase Complementation Assay to Identify Specific Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21332, doi: (2023).