实验概要
We provide a detailed protocol for antibody crosslinking to beads enabling elution of the target protein without contamination by the antibody.
主要试剂
1. Cross linking reagent:
Dimethyl pimelimidate (DMP)
Stock concentration 13 mg/ml DMP.
Working solution should be between pH 8 and pH 9.
2. Elution reagent:
1 M glycine (Add conc. HCl to correct pH to pH 3)
3. Dilution buffer:
PBS 1 mg/ml BSA
4. Wash buffer:
0.2 M triethanolamine in PBS (3.04 ml triethanolamine per 100 ml buffer)
5. Quenching buffer:
50 mM ethanolamine in PBS (311.7 μl per 100 ml)
实验步骤
1. Wash beads twice in PBS.
The end concentration should be 50 % bead slurry.
2. Mix well and rotate overnight at 4°C.
3. Wash the beads (protein A or protein G) by centrifuging (14,000 rpm, 1 min) into a pellet. Aspirate out the PBS supernatant.
4. Add dilution buffer at 1:1 ratio, mix gently and rotate for 10 minutes at 4°C. Centrifuge and aspirate/discard the supernatant as before.
5. 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.
6. Centrifuge and aspirate/discard the supernatant.
7. Add dilution buffer to beads at 1:1 ratio. Rotate for 5 min at 4°C. Centrifuge and aspirate/discard the supernatant.
8. Add PBS to beads at 1:1 ratio. Centrifuge and aspirate/discard the supernatant.
9. Cross-linking:
DMP is unstable in aqueous solution. Prepare solution immediately prior to use. |
Dissolve 1ml 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 8-9 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.
10. Quench and wash.
Add quench buffer at 1:1 ratio, rotate 5 min RT, spin and aspirate; repeat.
Wash with PBS.
11. Remove excess (unlinked) antibody:
Wash with 1 M glycine pH 3. Rotate 10 min RT. Repeat.
12. 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.
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