The overall goal of this procedure is to study protein-protein interactions in an intracellular context. This is achieved through a biotin-avidin pull-down system combined with self-penetrating sequences, allowing incubation at the target sequence with living cells. This method can help answer key questions in the cell signaling field such as the rapid and dynamic interactions between biomolecules to get by specific signals.
The main advantage of this technique is that the molecular interactions take place in an intracellular context. In this study, plate human G166 glioma stem cells or GSCs in four flasks of 150 square centimeters as described in the text protocol. Process the cells when confluence is reached.
When five million G166 cells are plated in a 150 square centimeter flask, they are processed two days after plating. Spin the vials containing the lyophilized biotinylated cell-penetrating peptides at 8, 200 times g for 30 seconds to avoid some of the powder remaining on the lid. Include a controlled BCPP to be sure that the interactions found are specific for the target sequence.
Then, dissolve the BCPPs in the corresponding culture medium to the stock solution indicated by the manufacturer. To obtain a stock solution of two milligrams per milliliter BCPP to treat GSCs, add 0.5 milliliters of GSC culture medium to one vial containing one milligram of the BCPP. Vortex the vials and ensure the peptide is well dissolved.
Prepare at least 12 1.5 milliliter tubes per condition in the pull-down assay to be performed. Mark the first three tubes per condition. They will have the total volume of cellular lysates.
Next, mark an A on three tubes per condition. These tubes will have the first supernatants obtained after lysing and spinning the cellular lysates. Then, mark a B on three tubes per condition.
These tubes will have a small aliquot of the first supernatants for western blot samples. Finally, mark a C on three tubes per condition. These tubes will have the supernatants obtained after the pull-down with NeutrAvidin.
After aspirating the culture medium from the cells, replace with 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 completely covers the whole surface of the flask. Add the volume of the stock solution of BCPP to the cell cultures to reach the concentration that has been proved to be effective.
Therefore, add 92.8 microliters of two milligrams per milliliter of TAT-connexin 43 266-283 biotin per milliliter of culture medium. Place the cells in the incubator at 37 degrees Celsius and 5%carbon dioxide for 30 minutes to make sure that the interactions between the BCPP and its intracellular partners take place. To perform the protein extraction, first aspirate the culture medium completely, then wash the cells with 10 milliliters of ice-cold phosphate buffered saline per 150 centimeters very carefully to avoid self-detachment.
To obtain the cell lysate, add three milliliters of lysis buffer per 150 square centimeter flask and thoroughly scrape the surface by using a cell scraper. Tilting the flask to about 45 degrees will make easier to gather the cell lysates into their corresponding tubes. Pour one milliliter of the cellular lysate per tube into three 1.5 milliliter tubes marked per condition.
For the pull-down, centrifuge the 1.5 milliliter tubes at 11, 000 times g for 10 minutes at four degrees Celsius. Transfer the supernatants to new tubes labeled A.Transfer an aliquot of the supernatant per condition to different tubes labeled B.Then, add 16.6 microliters of 4x Laemmli buffer for 50 microliters of lysate and freeze at minus 20 degrees Celsius. Next, homogenize the NeutrAvidin agarose very well by gentle shaking.
Cut the tips of the pipette tips to increase their diameter and improve the pipetting of the beads. Then, add 50 microliters of NeutrAvidin agarose per milliliter of cell lysate in the A tubes. Incubate the cell lysates at four degrees Celsius overnight with very gentle shaking to allow NeutrAvidin to interact with BCPPs bound to their intracellular partners.
Then, centrifuge at 3, 000 times g for one minute at four degrees Celsius to collect the complex of NeutrAvidin with proteins. Carefully remove the supernatants and transfer them to clean tubes labeled C.Keep them to use in case the pull-down is not successful. To wash the complex of NeutrAvidin with proteins, add fresh lysis buffer to the pellets of A tubes.
Resuspend the pellet by inversion and repeat the centrifugation. Repeat this wash for five more times. All these supernatants can be discarded.
Next, add 40 microliters of 2x Laemmli buffer per 1.5 milliliter tube for pellets obtained from 150 square centimeter flasks of confluent cells. Then, elute at 100 degrees Celsius for five minutes to dissociate the interactions between proteins. Following elution, centrifuge for 30 seconds at 8, 200 times g to pellet NeutrAvidin beads.
Transfer the supernatants containing the dissociated proteins with capillary tips to new tubes labeled D.These supernatants are now ready to be loaded in a western blot as described in the text protocol or can be frozen at minus 20 degrees Celsius. Shown here are immunocytochemistry images displaying that the fusion of biotin to TAT-connexin 43 266-283 does not modify the effects of the cell-penetrating peptide on glioma stem cell morphology. The MTT analysis of GSC treated with BCPP or CPP shows that the fusion of biotin to TAT-connexin 43 266-283 does not modify the anti-proliferative effect of TAT-connexin 43 266-283 on GSC.
Representative results of a BCPP pull-down followed by western blot are shown here. Western blot shows that proteins described to interact with TAT, for example, Cav-1, appear in both conditions. Proteins described to interact with Cx43, for example, c-Src and PTEN, appear just after TAT-connexin 43 266-283 pull-down, while proteins that do not directly interact with Cx43 or TAT are not present, for example, FAK.
Confocal microscopy images confirm the intracellular interactions found with the BCPPs. The images shows the intracellular colocalization of TAT-connexin 43 266-283 with c-Src close to the plasma membrane in G166 GSCs. Once mastered, this technique can be done in two days if it's performed properly.
While attempting this procedure, it's important to confirm the temperature of the reagents and the centrifuge as well as the confluence of the cells before adding the peptides. After watching this video, you should have a good understanding of how to study intracellular protein-protein interactions using biotinylated cell-penetrating peptides. After its development, this technique paves the way for researches in the field of cell signaling to explore intracellular molecular mechanisms and their line specific pathways in culture cells, organotypic cultures and even in vivo models.
Following this procedure, other bioactive cargoes such as RNA sequences, nanoparticles, viruses or other molecules that can transfused with cell-penetrating peptides and fused to pull-down TATs can be used to study their intracellular mechanism of action. Although no extremely hazardous materials are included in this protocol, don't forget to wear gloves and lab coat while performing this procedure.
