Tandem Affinity Purification Assay to Study Protein-Protein Interactions
Published: May 31, 2023
Abstract
Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification — a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.
Protocol
1. Plating Cells One day prior to transfection, plate 3.5 x 106 HEK293T cells in 10 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/Streptomycin (10,000 U/ml stock) per 100 mm dish. Plate 3 – 5 100 mm2 dishes per experiment/condition to ensure sufficient protein recovery. NOTE: The number of plates has to be determined for each experiment/condition, which will rely on the expression lev…
Representative Results
Table 1: Plasmids used in this Study. CycT1 and CDK9 were ligated into the pcDNA/4TO-STREP and pcDNA/4TO-FLAG plasmid vectors. These constructs were transformed into DH5α competent cells and plated on LB-Ampicillin plates. Colonies were selected, grown, and screened. Successfully ligated clones were verified by Sanger sequencing and the combination of restriction digestion and agarose gel electrophoresis. For CycT1 we used a shorter version (1 – 708) instead of the full-length (1 -…
Divulgazioni
The authors have nothing to disclose.
Materials
| Dulbecco's Modified Eagle Medium (DMEM) | Fisher Scientific | SH30022FS | |
| Fetal bovine serum | Hyclone | SH30071 | |
| Penicillin/Streptomycin | Fisher Scientific | MP091670049 | |
| PolyJet | Fisher Scientific | 50-478-8 | |
| Protease inhibitor cocktail | Roche | 11836153001 | |
| STREP-Tactin Superflow | IBA | 2-1208-010 | |
| STREP-tag elution buffer | IBA | 2-1000-025 | |
| EZview Red ANTI-FLAG M2 Affinity Gel | Sigma | SLBQ1981V | |
| Corning 100mm × 20 mm style Dish cell culture treated nonpyrogenic polystyrene 20/sleeve | Corning | 430167 | |
| Protein Lo-Bind Eppendorf | Fisher Scientific | 13-698-794 | |
| Digital Vortex Mixer | Fisher Scientific | 02-215-370 | |
| 48-hole micro tube foam rack | Fisher Scientific | 02-215-386 | |
| Labquake shaker rotisserie | Thermo | 415110 |
Tags
Citazione di questo articolo
Tandem Affinity Purification Assay to Study Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21405, doi: (2023).