Flag-tagged protein affinity purification and western blotting

Immunoprecipitation (IP) of Flag-tagged proteins was carried out with anti-Flag M2 magnetic beads as described³⁶. See Table ^S4 for the key biochemical resource summary. For small-scale experiments, a 50–100 ml culture was grown at 30 °C to exponential phase of 0.6. The culture was then transferred into 50 ml Falcon tube and centrifuged at 5500×g for 5 min, 4 °C. The cells were washed in ice-cold IP lysis buffer (30 mM HEPES–KOH pH 7.5, 10% (w/v) glycerol, 1 mM TCEP-HCl pH 7.0) with salt concentration adjusted to either 100 mM or 1 M KCl and Pierce protease inhibitor tablet added just before use). Washed cells were pelleted at 5500×g for 5 min, 4 °C and resuspended in 1 ml and lysed with 500 µl of acid washed glass beads at 6 × 20 s. Extract was separated from beads and debris by centrifugation at maximum speed (16,100×g) for 4 min and transfer into a fresh tube before a second centrifugation at maximum speed for 20 min, 4 °C. 1 mg of the total cell extracts of each strain were diluted in 500 µl IP lysis buffer and mixed with 50 µl of Flag magnetic resin. This was incubated for 1–2 h on a tube rotator set at 30 rpm, 4 °C. The bound proteins were washed four times; two washes in IP lysis buffer and two washes in IP_low buffer (10 mM HEPES–KOH pH 7.5, 100 mM KCl, 10% (w/v) glycerol, 1 mM TCEP-HCl pH 7.0). The bound proteins were eluted from the beads in 50 µl of 200 µg/ml of 3X Flag peptide. SDS-PAGE and western blotting were performed using reagents indicated in Table ^S4.

Large cultures (2 L) were grown in flasks as above, harvested by centrifugation and lysed 3 × 2 min under liquid nitrogen using a 6870 Freezer Mill (SPEX SamplePrep). For large scale IP for mass spectrometry analysis, for every 1 g of cryogenic ground cell, 2 ml of IP lysis buffer was added. Once thawed on ice, tubes were centrifuged for 10 min at 5500×g, 4 °C. The lysate was further clarified by centrifugation in 5 ml Eppendorf tubes at 16,100×g for 20 min. The protein concentration was measured and processed accordingly. A pre-clearing step incubating extracts with Sepharose 4B agarose beads (Sigma) before Flag magnetic beads reduced non-specific binding.