Biotinylated Cell-Penetrating Peptide Probe to Detect Protein-Protein Interactions
筆記録
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 2 milligrams per milliliter of TAT-Connexin-43266-283-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 cell detachment.
To obtain the cell lysate, add 3 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 1 milliliter of the cellular lysate per tube into 3 1.5-milliliter tubes marked per condition. For the pull-down, centrifuge the 1.5-milliliter tubes at 11,000 x g for 10 minutes at 4 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 4 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 1 minute at 4% 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 5 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 it can be frozen at minus 20 degrees Celsius.
