Materials and Reagents

HeLa S3 and HeLa cells were cultured in Dulbecco’s modified Eagles Medium containing 10% fetal bovine serum (Invitrogen) and antibiotics
Protein A beads (Bio-Rad Laboratories); Protein G beads (Santa Cruz Biotechnology)
Note: For rabbit polyclonal antibodies and mouse monoclonal antibodies IgG2a, IgG2b, IgG3, we usually couple antibodies to protein A beads; For mouse monoclonal antibodies IgG1, rat monoclonal antibodies, and mouse, rat, goat, and donkey polyclonal antibodies, we usually couple antibodies to protein G beads.
Bifunctional cross-linker dimethyl pimelimidate (DMP) (MW: 259.177) (Sigma-Aldrich, catalog number: D8388 )
Phosphate buffered saline (PBS)
Tween 20
Triton X-100
Sodium borate (pH 9.0)
Hepes
KCl
NaN₃
NP-40
Glycerol
EGTA
MgCl₂
DTT
Microcystin
Leupeptin
Pepstatin
Chymostatin
β-mercaptoethanol
Bromophenol blue
PBST buffer (see Recipes)
Lysis buffer (see Recipes)
1x SDS protein gel sample loading buffer (see Recipes)

Equipment

Hematology/chemistry mixer (Fisher Scientific)
Centrifuge (Eppendorf 5415D centrifuge)
Incubator

Procedure

Coupling antibody to Protein A beads
1. Wash 30 μl beads with 20 fold volume (20 vol) PBS or PBST twice.
  Notes:
  1. For washing beads, we usually add buffer to the beads, mix in the tube several times, spin down the beads, and remove the supernatant.
  2. Spin down the beads at 4,000 rpm, 30 sec.
  3. We usually couple 1 μg antibody with 3 μl beads. If beads are stored in 1: 1 storing buffer, thus take 6 μl of the suspension of beads and buffer.
2. Dilute 10 μg antibody in 100 μl PBS.
3. Add diluted antibody to beads, rotate on the Hematology/chemistry mixer equipment for 1 h at room temperature (RT).
4. Spin down beads, wash and resuspend in 20 vol 0.2 M sodium borate (pH 9.0).
5. Add 20 mM DMP to crosslink the antibody to the beads.
  Note: We usually take dry DMP powder directly (DMP powder is stored at 4 °C) and add 5.2 mg DMP per 1 ml total sodium borate suspension.
6. Incubate DMP in the sodium borate for 30 min, rotate, RT.
7. Spin down the bead and wash once with 20 vol 1 M Tris-HCl (pH 7.7).
8. Spin down the bead and resuspend in 20 vol 1 M Tris-HCl (pH 7.7). Incubate for 2 h, rotate, RT, to quench the activity of DMP.
9. Spin down beads and wash with PBS twice.
10. Spin down the beads and store antibody coupled beads in 60 μl PBS (33% beads), add 0.05% NaN₃. Store the beads at 4 °C for a couple of months.
Lysis of cells
1. Lyse the cells in 7 vol lysis buffer.
2. Put the lysis solution on ice for 30 min to 1 h.
3. Centrifuge the lysis solution at 13,000 rpm for 10 min, 4 °C.
4. Transfer supernatants. The supernatants can be stores at -80 °C for future use.
Coimmunoprecipitation
1. Incubate 3 μl antibody coupled beads in 250 μl cell lysis supernatants, rotate overnight at 4 °C.
2. Wash beads 4 times with 20 vol lysis buffer containing 500 mM KCl and 0.5 % NP-40, and wash once with lysis buffer.
  Note: For wash beads, we usually add buffer to the beads, mix the tube several times, spin down the beads, and remove the supernatant.
3. Elute protein with 1x SDS protein gel sample loading buffer, separated by SDS-PAGE (5-15%) and analyze by western blot.

Recipes

PBST buffer
PBS plus 0.1 % Tween 20 or 0.1 % Triton X-100
Lysis buffer
50 mM Hepes (pH 7.4)
200 mM KCl
0.3% NP-40
10% glycerol
1 mM EGTA
1 mM MgCl₂
0.5 mM DTT
0.5 μM microcystin
10 μg ml^-1each of leupeptin, pepstatin, and chymostatin
1x SDS protein gel sample loading buffer
50 mM Tris-HCl (pH 6.8)
2% SDS
10% glycerol
1% β-mercaptoethanol
12.5 mM EDTA
0.02 % bromophenol blue

Acknowledgments

This protocol was modified from an immunoprecipitation protocol developed in the laboratory of Dr. Guowei Fang (Department of Biology, Stanford University, Stanford, CA, USA). This work was supported by a Burroughs-Wellcome Career Award in Biomedical Research (G.F.) and by grants from National Institutes of Health (GM062852 to G.F.).

References

Zhu, H., Coppinger, J. A., Jang, C. Y., Yates, J. R., 3rd and Fang, G. (2008). FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. J Cell Biol 183(5): 835-848.