Phosphopeptide Enrichment: An Antibody-based Immunoprecipitation Technique to Separate Specific Phosphorylated Peptides from Complex Peptide Mixtures

Resuspend the lyophilized powder with 0.5 milliliters of ice-cold immunoprecipitation, or IP, binding buffer in each fraction. Transfer the 0.5 milliliter resuspension volume from the second fraction to the tube with the first fraction and save the pipette tip. Use another 0.5 milliliters of IP buffer to rinse each lyophilization tube before transferring the contents to the cryotube. Then, repeat the rinse using 2 milliliters of IP buffer.

After using P200 with a cut tip to transfer the 0.5 milligrams per milliliter stock of antibody bead slurry to the microfuge tube, add 450 microliters of ice-cold IP binding buffer to wash 50 microliters of 4G10 antibody bead slurry and 25 microliters of 27B10.4 antibody bead slurry in a microfuge tube. Centrifuge the tube at 100 x g and 4 degrees Celsius for 1 minute. After aspirating the supernatant, add an equal volume as the original slurry of IP binding buffer to resuspend the beads. Add pre-washed pY beads to the resuspended sample solution in the screw cap cryotubes. Then, incubate them at 4 degrees Celsius on an end-over-end rotator overnight. After spinning down the beads, save the supernatant, which will be used to enrich for pST peptides.

Next, use 300 microliters of IP binding buffer to resuspend the beads and transfer them to a 2-milliliter microcentrifuge tube. Spin down the samples at 100 x g and 4 degrees Celsius for 1 minute. Then, with 500 microliters of IP binding buffer, wash the beads three times. Wash the beads four times with 450 microliters of 25 mM ammonium bicarbonate, pH 7.5. Dip a gel-loading tip slightly below the bead surface and completely aspirate the supernatant. Add four times the bead volume of 0.1% TFA to the beads.

Then, mix them well and incubate the tube in a thermomixer at 1,000 rpm and 37 degrees Celsius for 15 minutes. Transfer the resuspension to a 0.2-micrometer spin filter, and centrifuge the filter at 850 x g for 1 minute. After transferring the elution to a low protein-binding microcentrifuge tube, vacuum concentrate the eluate to dryness at 40 degrees Celsius with a heat time of 300 minutes overnight.