Protein low bind microcentrifuge tubes

Bacteria were resuspended in 150 μL lysis buffer (1% Triton X-100, 0.5% SDS, 1 tablet cOmplete EDTA-free protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) in 10 mL PBS) and lysed by ultrasonication (5 × 20 s, 80%, on ice during breaks). Cell debris was pelletized (5000g, 10 min, 4 °C) and the supernatant was sterile filtered through a 0.2 μm pore size PTFE filter. Protein concentration was determined using a BCA assay (Roti-Quant universal, Carl Roth GmbH + Co. KG), all samples were adjusted to the same volume and concentration (ca. 1 mg mL⁻¹) and transferred to protein low-bind microcentrifuge tubes (Eppendorf, Hamburg, Germany). To precipitate the proteins, 4× sample volume acetone (−80 °C) was added and the samples were stored at −80 °C overnight. The samples were centrifuged at 21 000g at 4 °C for 15 min and the supernatant was discarded. The pellet was resuspended in 500 μL methanol (−80 °C) with ultrasonication (10 s, 10%). After centrifugation at 21 000g and at 4 °C for 15 min, the supernatant was discarded and the pellet was air-dried.

Bacteria were resuspended in 800 μL co-IP lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 5% glycerol, pH 7.4) and 120 μg lysozyme was added. The samples were incubated at 37 °C under shaking at 1400 rpm for 1 h. Afterwards, 8 μL 10% NP-40 solution was added and the bacteria were lysed by ultrasonication (5 × 30 s, 80%, on ice during breaks). The insoluble fraction was pelletized (10 000g, 30 min, 4 °C) and the supernatant was sterile filtered through a 0.2 μm PTFE filter. Protein concentration was determined using a BCA assay (Roti-Quant universal, Carl Roth GmbH + Co. KG). 30 μL Protein A/G agarose beads (Thermo Fisher Scientific) were transferred to protein low-bind microcentrifuge tubes (Eppendorf) and washed with 1 mL co-IP wash buffer (50 mM Tris–HCl, 150 mM NaCl, 5% glycerol, 0.05% NP-40, pH 7.4) and centrifuged for 1 min at 1000g at 4 °C. 500 μg proteome (in 500 μL) and either 1 μL anti-c-Myc antibody (rabbit polyclonal, ab152146, 1 mg mL⁻¹, Abcam) or 0.4 μL rabbit mAb IgG isotype control (2.5 mg mL⁻¹, Cell Signaling Technology, Danvers, United States) were added. The samples were incubated at 4 °C for 3 h under constant rotation. The supernatant was removed after centrifugation (1000g, 1 min, 4 °C), and the beads were washed twice with 1 mL co-IP wash buffer. The detergent was removed by washing the beads twice with co-IP lysis buffer.