Bimolecular Complementation Affinity Purification to Isolate Two Interacting Proteins
Transcript
First, seed HEK293T cells in 10-centimeter dishes containing 10 milliliters of DMEM. Then, dilute 2.5 micrograms of each bimolecular fluorescence complementation vector in 500 microliters of transfection buffer.
Add 10 microliters of the transfection reagent, and vortex the mixture for 10 seconds. Then, briefly centrifuge the samples, and incubate them at room temperature for 10 minutes. Next, add the DNA transfection mixture drop-wise to the dish. Then, incubate the samples for 8 to 24 hours.
Prepare cell lysis buffer and supplemented cell lysis buffer as outlined in the text protocol. Then, wash the cells with ice-cold PBS twice. Aspirate the PBS, and add 1 milliliter of ice-cold supplemented cell lysis buffer.
Next, place the dish on ice, and incubate for five minutes. Then, use a cell scraper to remove the cells and transfer them to a chilled microcentrifuge tube. Centrifuge the tube at 18,000 times gravity for five minutes at 4 degrees Celsius to remove the cellular debris. Then, transfer the clear supernatant to a fresh microcentrifuge tube.
Wash an appropriate volume of agarose beads in 1 milliliter of PBS. Then, centrifuge the beads at 300 times gravity, and carefully remove the supernatant. Next, add 20 microliters of agarose beads to each sample, and incubate at 4 degrees Celsius for two hours with end-to-end rotation.
Centrifuge the beads at 300 times gravity, and wash them in cell lysis buffer three times. Then, resuspend the washed beads in 50 microliters of diluted sample buffer. Finally, heat the samples at 95 degrees Celsius for two to three minutes.
