To enable elution of protein with little antibody contamination (for cleaner protein preparation and cleaner western blots), it is recommended to cross-link the antibody to the beads. An example procedure for this is shown below. The target protein should then be eluted with a mild eluent, such as glycine buffer.
Elution reagent: 1 M glycine (Add conc. HCl to correct pH to 3)
Dilution buffer: 1mg/mLBSA in PBS
Wash buffer: 0.2 M triethanolamine in PBS (3.04 ml triethanolamine per 100 ml buffer)
Quenching buffer: 50 mM ethanolamine in PBS (311.7 μL per 100 mL)
Wash beads twice in PBS. The end concentration should be 50% bead slurry.
Mix well and rotate overnight at 4°C.
Wash the beads (protein A or protein G) by centrifuging (14,000 rpm, 1 min) into a pellet. Aspirate out the PBS supernatant.
Add dilution buffer at 1:1 ratio (eg for 50 µL of beads add 50 µL of dilution buffer), mix gently and rotate for 10 min at 4°C. Centrifuge and aspirate/discard the supernatant as before.
Prepare the antibody solution in dilution buffer at the required concentration (see antibody datasheet for suggested concentration). Add diluted antibody at 1:1 ratio to the beads. Mix gently and rotate 1 hr at 4°C.
Centrifuge and aspirate/discard the supernatant.
Add dilution buffer to beads at 1:1 ratio. Rotate for 5 min at 4°C. Centrifuge and aspirate/discard the supernatant.
Add PBS to beads at 1:1 ratio. Centrifuge and aspirate/discard the supernatant.
DMP is unstable in aqueous solution. Prepare solution immediately prior to use. Dissolve 1 ml of prepared 13 mg/ml stock of DMP with 1 ml wash buffer. Vortex immediately to mix. Add DMP solution to beads at 1:1 ratio. Rotate for 30 min at room temperature.
N.B. You will need to verify pH of DMP is between 8 and 9 before and after addition to beads (cross-linking efficiency is greatly reduced outside this pH range). Wash the beads with wash buffer (rotate 5 min RT, then spin and aspirate). Add DMP for second time at 1:1 ratio, rotate 30 min RT, wash as before. Add DMP for third time at 1:1 ratio, rotate 30 min RT, wash as before.
Quench and wash. Add quench buffer at 1:1 ratio, rotate 5 min RT, spin and aspirate; repeat. Wash with PBS.
Remove excess (unlinked) antibody: Wash with 1 M glycine pH 3. Rotate 10 min RT. Repeat.
Storage washes. Wash with buffer to be used for immunoprecipitation (usually PBS+TWEEN). Rotate 5 min RT. Wash three times and store in final wash (after rotation). Beads can be stored at 4°C for a few days. Sodium azide can be added to prevent bacterial growth.
The antibody-bound beads can now be used in a normal IP procedure. Elution of bound antigens: To prevent elution of antibody with the target protein, use a gentle glycine elution gradient (up to 1 M).