Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 308

background although there is also an increased likelihood of losing meaningful low
affinity interactions.
1. Pre-elute 100 m L of antibody-coupled beads three times with 1 mL of
100 mM glycine (pH 2.6) to remove antibody that is not covalently coupled
to the beads. Do not leave beads for a long time in glycine or the antibody will
denature.
2. Wash beads three times with 1 mL cold Lysis Buffer (with 0.5 mM DTT) to
neutralize glycine and prepare the beads for immunoprecipitation.
3. Mix beads with 900 m L extract for at least 1 h at 4 C on rotating platform.
4. Rinse beads two times with 1 mL cold Lysis Buffer (with 0.5 mM DTT and
protease inhibitors).
5. On rotator in cold room wash beads two times for 5 min with 1 mL cold Lysis
Buffer (with 0.5 mM DTT and protease inhibitors).
6. Wash five times with 1 mL Lysis Buffer (with 0.5 mM DTT) without detergent
(NP-40) or protease inhibitors. Remove as much supernatant as possible.
Note: The presence of detergents can interfere with mass spectrometry. Therefore,
it is important to wash the sample thoroughly in detergent-free buffer after
immunoprecipitation.
C. Sample Buffer Elution: For Silver Staining & Immunoblotting
1. Elute beads by heating in 100 m Lof2 Sample Buffer without DTT for 10 min
at 70 C.
2. Pellet beads, transfer supernatant to a new tube, and add DTT to 100 mM (1/9
supernatant volume of 1 M DTT stock; Elution 1).
3. Add 100 m L2 Sample Buffer with DTT to pelleted beads (Elution 2).
4. Boil both elution samples for 5 min and analyze by silver staining or immunoblot.
Both elutions will contain immunoprecipitated proteins although amounts in
each may vary; Elution 2 will have more IgG contamination than Elution 1.
Note: For immunoblots conducted using rabbit primary antibodies, the secondary
antibody will detect any IgG released by the elution from the beads. For silver
staining, load 5-10 m L directly. For immunoblots, load 5-10 m L of a 1/10 dilution
made in Sample Buffer.
D. Glycine Elution: For Mass Spectrometry
1. After standard immunoprecipitation (see Section VI.B), elute beads three times
with 150 m L 100 mM glycine (pH 2.6). Pool elutions and neutralize by adding
150 m L 2 M Tris (pH 8.5). Neutralize the beads by washing two times with
150 m L Lysis Buffer (without detergent) and pool with eluate. Total volume will
be 900 m L. Make sure you remove all the beads.