All tags Immunoprecipitation Cross-linking antibodies to beads protocol

Cross-linking antibodies to beads protocol

Detailed procedure for antibody cross-linking to beads enabling elution of the target protein without contamination by the antibody.

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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.

Reagents:

Cross linking reagent:
Dimethyl pimelimidate (DMP)
Stock concentration 13 mg/ml DMP.

Working solution should be between pH 8 and pH 9.

Elution reagent:
1 M glycine (Add conc. HCl to correct pH to 3)

Dilution buffer:
PBS + 1 mg/ml BSA

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)

Preparation:

  1. Wash beads twice in PBS.
    The end concentration should be 50% bead slurry.
  2. Mix well and rotate overnight at 4°C.

Cross-linking:

  1. Wash the beads (protein A or protein G) by centrifuging (14,000 rpm, 1 min) into a pellet. Aspirate out the PBS supernatant.
  2. 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.
  3. 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.
  4. Centrifuge and aspirate/discard the supernatant.
  5. Add dilution buffer to beads at 1:1 ratio. Rotate for 5 min at 4°C. Centrifuge and aspirate/discard the supernatant.
  6. Add PBS to beads at 1:1 ratio. Centrifuge and aspirate/discard the supernatant.
  7. Cross-linking:


    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.
  8. Quench and wash.
    Add quench buffer at 1:1 ratio, rotate 5 min RT, spin and aspirate; repeat.
    Wash with PBS.
  9. Remove excess (unlinked) antibody:
    Wash with 1 M glycine pH 3. Rotate 10 min RT. Repeat.
  10. 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.

Immunoprecipitation:

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).

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