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

Overview

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

1. 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.
2. In this study, plate human G166 GSCs in 4 flasks of 150 cm², cultured in RHB-A stem cell medium supplemented with 2% B27, 1% N2, 20 ng/mL EGF, and b-FGF as described by Pollard et al. Process when confluence was reached. For instance, when 5 x 10⁶ G166 cells were plated in a 150 cm² flask, they were processed 2 days after plating.

2. Biotinylated CPPs

1. Spin the vials containing the lyophilized biotinylated CPPs (BCPPs) at 8200 x g for 30 s, to avoid some of the powder remaining on the lid. Include a control BCPP to be sure that the interactions found are specific to the target sequence. In this study, the control BCPP was TATbiotin (TAT-B), and the treatment BCPP was TAT-Cx43_266-283-B. Other controls could be used, such as TAT fused to scrambled or mutated fragments bonded to biotin.
2. Dissolve the BCPPs in the corresponding culture medium to the stock solution indicated by the manufacturer; for instance, to obtain a stock solution of 2 mg/mL BCPP to treat GSCs add 0.5 mL of GSC culture medium to one vial containing 1 mg of the BCPP. Vortex and make sure the peptide is well-dissolved.

3. Tubes

NOTE: Prepare at least twelve 1.5 mL tubes per condition required in the Section 

1. Mark the first 3 tubes per condition. They will have the total volume of cellular lysates obtained in step 6.4.
2. Mark an A in 3 tubes per condition. These tubes will have the first supernatants obtained after lysing and spinning the cellular lysates, step 7.2.
3. Mark a B in 3 tubes per condition. These tubes will have a small aliquot of the first supernatants. These lysates will serve as the Western blot samples in step 7.3.
4. Mark a C in 3 tubes per condition. These tubes will have the supernatants obtained after the pull-down with NeutrAvidin, step 7.7.
   NOTE: This is important in case the Western blot showed no signal for proteins, meaning the proteins were not pulled down or were lost at some step of the procedure. If this were the situation, repeat the process to pull down the proteins.

4. Cellular treatment with the BCPPs

1. Aspirate the culture medium.
2. Replace the corresponding volume of fresh medium required to incubate the BCPPs in the smallest possible volume of medium according to the incubation times. It is very important that in any case, the medium covers completely the whole surface of the plate/flask so that the cells do not dry out, for instance, 6 mL per 150 cm² for a 30 min incubation.
3. Add the volume of the stock solution of BCPP to the cell cultures to reach the concentration that has been proven to be effective. In this study, 50 µM TAT-Cx43266-283-B has been proven to reduce GSC proliferation.
   1. Therefore, add 92.8 µL of 2 mg/mL TAT-Cx43_266-283-B (MW=3723.34 g/mol) per mL of culture medium to obtain a final concentration of 50 µM TAT-Cx43_266-283-B. If the volume to be added is different for control peptides, complete with culture medium up to the same final volume. For instance, 49.1 µL TAT-B (MW=1914.31 g/mol) plus 43.7 µL GSC medium were added per mL of culture medium to obtain a final concentration of 50 µM TAT-B.
4. Place the cells in the incubator at 37 °C and 5% CO₂ for 30 min to make sure that the interactions between the BCPP and its intracellular partners take place. If the interaction takes longer, incubate for longer times or adjust times in case the experiment consisted of a time course. In addition, the interaction of interest can be promoted or prevented by stimulating different intracellular signaling pathways.

5. Buffers and solutions

1. Prepare PBS pH 7.4: 136 mM NaCl; 2.7 mM KCl; 7.8 mM Na₂HPO₄·2H₂O; 1.7 mM KH₂PO₄.
2. Prepare protein lysis buffer: 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% IGEPAL, Prior to use, add the following: 1/100 (v/v) Protease Inhibitor Cocktail, 1 mM Sodium Fluoride, 1 mM Phenylmethanesulfonyl fluoride (PMSF) and 0.1 mM Sodium orthovanadate.
3. Prepare Laemmli buffer: (4x: 0.18 M Tris-HCl pH 6.8; 5 M glycerol; 3.7 % SDS (p/v); 0.6 M β-mercaptoethanol or 9 mM DTT; 0.04 % (v/v) bromophenol blue (BB)).

6. Protein extraction

NOTE: Protein extraction was performed as previously described. Carry out this whole section of the procedure at 4 °C.

1. Aspirate the culture medium completely.
2. Wash 3 x 10 mL with ice-cold phosphate-buffered saline (PBS) per 150 cm² very carefully to avoid cell detachment.
3. To obtain the cell lysate, add 3 mL of lysis buffer per 150 cm² and thoroughly scrape the surface by using a cell scraper. Tilting the plate/flask to about 45 degrees will make it easier to gather the cell lysates into their corresponding tubes.
4. Pour 1 mL of the cellular lysate per tube into three 1.5 mL tubes. These tubes will be marked with the condition and the replicate as indicated in step 3.1.

7. Pull-down

1. Centrifuge the 1.5 mL tubes at 11,000 x g for 10 min at 4 °C.
2. Transfer the supernatants to new tubes (A).
3. Take an aliquot of this supernatant per condition to different tubes (B), i.e. 50 µL per tube. Add 4x Laemmli buffer (16.6 µL for 50 µL of lysate) and freeze at -20 °C. These lysates will serve as usual Western blot samples.
4. Homogenize very well the NeutrAvidin Agarose by gentle shaking. Cut the tips of the pipette tips to increase their diameter and improve the pipetting of the beads. Add 50 µL of NeutrAvidin Agarose per mL of cell lysate in the (A) tubes.
5. Incubate at 4 °C overnight with very gentle shaking to allow NeutrAvidin to interact with BCPPs bound to their intracellular partners.
6. Centrifuge at 3,000 x g for 1 min at 4 °C to collect the complex of NeutrAvidin with proteins.
7. Carefully remove the supernatants and transfer them to clean tubes (C). Keep them to use them in case the pull-down is not successful.
8. Washing steps: add fresh lysis buffer to the pellets (tubes A), resuspend by inversion, and repeat steps 7.6 and 7.7 five more times. All these supernatants can be discarded.
9. Remove the supernatants and add 2x Laemmli buffer to the desired final volume (40 µL per 1.5 mL tube for pellets obtained from cm² flasks of confluent cells).
10. Elute at 100 °C for 5 min to dissociate the interactions between proteins and centrifuge for 30 s at 8,200 x g to pellet NeutrAvidin beads.
11. Take the supernatants, containing the dissociated proteins, with capillary tips to new tubes (D). The beads can be kept in case repeating elution steps is required.
    NOTE: The supernatants (tubes D) are now ready to be loaded in Western blot or to freeze at -20 °C.

Materials

Name	Company	Catalog Number	Comments
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) .