Cross linking reagent:
Dimethyl pimelimidate (DMP)
Stock concentration 13 mg/ml DMP.
Working solution should be between pH 8 and pH 9.
1 M glycine (Add conc. HCl to correct pH to 3)
PBS + 1 mg/ml BSA
0.2 M triethanolamine in PBS (3.04 ml triethanolamine per 100 ml 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, 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).
Print this protocol.