Tandem Affinity Purification Assay to Study Protein-Protein Interactions
Trascrizione
After the cell lysate and STREP-bead slurry mixtures have incubated for 2 hours, spin them down at 1,1500 times g for 2 minutes at 4 degrees Celsius. Save the supernatant as "STREP AP flow-through," and store at 4 degrees Celsius.
Add 500 microliters of STREP AP Wash Buffer 1 to the beads. Nutate the beads and buffer mixture for 5 minutes at 4 degrees Celsius, and centrifuge at 1,500 times g for 2 minutes at 4 degrees Celsius. Discard the supernatant, and wash again with 500 microliters of STREP AP Wash Buffer 1.
After the third wash, transfer the bead solution to a new 1.5-milliliter microcentrifuge tube to reduce the protein background. Wash a fourth time with STREP AP Wash Buffer 1. After the spin, remove the supernatant, and add 500 microliters of Wash Buffer 2.
Equilibrate the beads by inverting the tubes, and then, spin down at 1,500 times g for 2 minutes at 4 degrees Celsius. Discard the supernatant. Elute the protein of interest with 50 microliters of STREP-elution buffer. Vortex at 800 RPM for 15 minutes at 4 degrees Celsius. Spin down the beads at 1500 times g for 1 minute at 4 degrees Celsius. Collect the supernatant. This fraction corresponds to the "STREP AP elution" sample.
Save the beads fraction at 4 degrees Celsius for a second elution. Aliquot 10% of the STREP AP elution for silver staining and/or western blotting.
To increase the amount of starting material for the subsequent FLAG immunoprecipitation, the first and second STREP elutions can be pooled together for more efficient recovery of the FLAG elution.
Begin this procedure by using a wide-mouthed pipette tip to transfer the appropriate amount of FLAG bead solution to a microcentrifuge tube. Spin down at 1,500 times g for 2 minutes at 4 degrees Celsius. Remove the supernatant, and add 500 microliters of FLAG Wash Buffer to the beads.
Spin down at 1,500 times g for 2 minutes at 4 degrees Celsius. Discard the supernatant, and wash again with FLAG wash buffer. After the third wash, remove the supernatant, and resuspend the beads in FLAG wash buffer to a final volume of n times 16 microliters where n is the number of samples.
Add 16 microliters of the FLAG bead slurry to the remaining STREP AP elution, and nutate overnight at 4 degrees Celsius. Following an overnight incubation of the FLAG bead slurry and STREP AP elution, proceed with FLAG immunoprecipitation.
Spin down the FLAG beads suspension at 1,500 times g for 2 minutes at 4 degrees Celsius. Save the supernatant as the "FLAG IP flow-through," and store at 4 degrees Celsius.
Add 500 microliters of FLAG wash buffer to the beads. Nutate for 5 minutes at 4 degrees Celsius, and centrifuge at 1,500 times g for 2 minutes at 4 degrees Celsius. Discard the supernatant, and wash again with 500 microliters of FLAG wash buffer.
After the third wash, transfer the protein bead solution to a new 1.5-milliliter microcentrifuge tube to reduce background. Wash a fourth time with FLAG wash buffer. Remove the supernatant from the final spin, and add to the beads 30 microliters of FLAG wash buffer containing 200 nanograms per microliter of FLAG peptide. Vortex at 800 RPM for two hours at 4 degrees Celsius.
After two hours, spin down the suspension at 1,500 times g for 2 minutes at 4 degrees Celsius. Harvest the supernatant, and store at 4 degrees Celsius as the "FLAG IP elution."
