Nickel nitriloacetic acid agarose

For purification of recombinant proteins from E. coli, cells were grown in 1l LB medium at 37ºC to OD₆₀₀ of 0.4–0.7 and then cooled to 16°C, and protein expression was induced by the addition of 0.75 mM isopropyl β-d-1-thiogalactopyranoside. Cells were harvested after overnight growth and were washed once with 100 ml of cold Tris buffer (50 mM Tris, pH 7.4, and 150 mM NaCl) at 3500 × g for 10 min. The pellet was resuspended in 15 ml of buffer containing 0.05× PIC (protease inhibitor cocktail; 1x = 0.1 mg/ml of leupeptin, 1 mM o-phenanthroline, 0.5 mg/ml of pepstatin A, 0.1 mM Pefabloc) and 1 mM phenylmethylsulfonyl fluoride. Cells were lysed using the Microfluidizer M-110L (Microfluidics, Newton, MA), and the lysate was centrifuged for 30 min at 20,000 × g at 4°C. The cleared lysate was incubated with glutathione Sepharose 4B (GE Healthcare), nickel-nitriloacetic acid agarose (Qiagen GmBH, Hilden, Germany), or amylose resin (New England BioLabs, Frankfurt, Germany) for 2 h at 4ºC. The resin was washed with 20 column volumes of the buffer, and bound protein was eluted from the respective beads using 20 mM glutathione, 0.3 M imidazole, or 10 mM maltose. The eluted protein was desalted on a PD-10 column using storage buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5% glycerol), flash frozen, and stored at −80ºC in small aliquots.

For coimmunoprecipitations, we modified the foregoing immunoprecipitation protocol to use a smaller scale, starting with 50 ml of log-phase (OD₅₉₅ = 0.5) cells. For Figure 1C, nickel-nitriloacetic acid agarose (Qiagen) was used instead of anti-HA beads, and 10 mM imidazole was added in lysis buffer. Western blots were probed with anti-6His (SC-8036; Santa Cruz Biotechnology), anti-GFP (Moseley et al., 2009 (link)), and anti-myc (SC-40, Santa Cruz Biotechnology, Dallas, TX) antibodies.