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.
1. Cells
2. Biotinylated CPPs
3. Tubes
NOTE: Prepare at least twelve 1.5 mL tubes per condition required in the Section
4. Cellular treatment with the BCPPs
5. Buffers and solutions
6. Protein extraction
NOTE: Protein extraction was performed as previously described. Carry out this whole section of the procedure at 4 °C.
7. Pull-down
The authors have nothing to disclose.
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) . |