Biotinylated Cell-Penetrating Peptide Probe to Detect Protein-Protein Interactions

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…

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) .