Biotinylated Cell-Penetrating Peptide Probe to Detect Protein-Protein Interactions
Published: May 31, 2023
Abstract
Source: Jaraíz-Rodríguez, M. et al., Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions. J. Vis. Exp. (2017).
This video demonstrates a technique that uses biotinylated cell-penetrating peptides to detect intracellular protein-protein interactions. The cell-penetrating peptides facilitate the conjugated peptide to enter a cell and specifically bind to its interacting proteins. The biotin, after binding to avidin, allows purification of the interacting proteins’ complex from a cell lysate.
Protocol
1. Cells Two days before starting the procedure, plate the cells at the required density to be confluent on the day of the experiment. Plate the cells in flasks or plates. However, protein extraction will be easier from plates. It is convenient to prepare at least 4 plates of 78 cm2 or 2 flasks of 150 cm2 per condition per experiment, to be sure that the results are consistent. In this study, plate human G166 GSCs in 4 flasks of 150 cm2, cultured in RHB-A stem cell m…
開示
The authors have nothing to disclose.
Materials
| G166 GSC line | BioRep | ||
| RHB-A stem cell medium | Takara | Y40001 | |
| Laminin Mouse Protein | Invitrogen, Life Technologies, ThermoFisher Scientific | 23017-015 | 10 µg/ml |
| B-27 Serum-free Supplement (50X) | Invitrogen, Life Technologies, ThermoFisher Scientific | 17504-044 | 2% |
| N-2 Supplement (100x) | Invitrogen, Life Technologies, ThermoFisher Scientific | 17502-048 | 1% |
| Recombinant Human EGF | Peprotech | AF-100-15 | 20 ng/ml |
| Recombinant Human b-FGF | Peprotech | AF-100-18B | 20 ng/ml |
| PBS pH 7.4: In deionized water, 136 mM NaCl ; 2.7 mM KCl; 7.8 mM Na2HPO4·2H2O; 1.7 mM KH2PO4 | |||
| TAT | GenScript | Custom-made | |
| TAT-B | GenScript | Custom-made | |
| TAT-Cx43266-283 | GenScript | Custom-made | |
| TAT-Cx43266-283-B | GenScript | Custom-made | |
| Pierce™ NeutrAvidin™ Agarose | ThermoFisher Scientific | 29200 | |
| Protein lysis buffer: 5 mM Tris HCl (pH 6.8), 2% (w/v) SDS, 2 mM EDTA , 2 mM EGTA | |||
| Sodium Fluoride | PanReac AppliChem | 141675 | 1 mM |
| Phenylmethanesulfonyl fluoride (PMSF) | Sigma-Aldrich | P7626 | 1 mM |
| Sodium orthovanadate | Sigma-Aldrich | S6508 | 0.1 mM |
| Laemmli buffer: (4x: 0.18 M Tris-HCl pH 6.8; 5 M glycerol; 3.7 % (w/ v) SDS; 0.6 M β-mercaptoethanol or 9 mM DTT ; 0.04% (v/v) bromophenol blue (BB) . |
