Protein isolation and western blot

Protein was isolated from M. abscessus by pelleting bacteria 3200 × g for 10 minutes at 4°C, resuspending in Tris buffered saline (TBS) (28358 Thermo Fisher Scientific) + protease inhibitor (11873580001, MilliporeSigma) (0.5 tablet per 10 mL TBS), transferring to 2 mL tubes with 0.1 mm silica beads (116911500, MP Biomedicals), and homogenizing using a Bead Bug 3 Microtube Homogenizer 4 × 45 seconds at 4000 rpm with 2 minutes of incubation on ice between rounds of homogenization. Homogenized samples were pelleted 21,130 × g for 5 minutes at 4°C, and the supernatant was heat-killed by incubation at 80°C for 20 minutes. Protein abundance was quantitated by absorbance at 280 nm using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific), and samples were normalized to 2.5 mg/mL by dilution in TBS. After normalization, remaining DNA was digested by addition of TURBO DNase buffer (AM2238, Thermo Fisher Scientific) (final concentration of 10%) and TURBO DNase (final concentration of 2%) followed by incubation at 37°C for 15 minutes. Samples were mixed with 4X LDS NuPage sample buffer to a final concentration of 1X and dithiothreitol (71003-396, VWR) to a final concentration of 50 mM. Samples were incubated at 70°C for 10 minutes, then 7 μg of protein along with PageRuler Prestained ladder 10 kDa to 180 kDa (26616, Thermo Fisher Scientific) was loaded on a NuPage 4–12% gradient Bis-Tris pre-cast SDS-PAGE gel (NP0321, Thermo Fisher Scientific), which was electrophoresed at 115 V for 90 minutes. Proteins were transferred to a PVDF membrane (1704156, Bio-Rad Laboratories, Hercules, CA) using TransBlot Turbo Transfer System (Bio-Rad) on the Mixed MW setting. Membranes were blocked by incubating in TBS + 0.1% Tween 20 (TBST) + 5% bovine serum albumin 1 hr at 22°C, and then were incubated with streptavidin-HRP (3999S, Cell Signaling Technology, Danvers, MA, USA) diluted 1:400,000 in TBST + 5% bovine serum albumin for 18 hr at 4°C. Membranes were washed 3x in TBST to remove unbound streptavidin-HRP and were developed by 1 minute incubation in 1 mL Azure Radiance Plus luminol/enhancer solution and 1 mL Azure Radiance Plus Peroxide Chemiluminescent Detection Reagent (AC2103, Azure Biosystems). Excess reagent was allowed to drain off of the membrane, and membranes were imaged using the chemiluminescence detector of a c300 Gel Imaging System (Azure Biosystems). After blotting, total protein was detected by staining membranes with SYPRO Ruby Protein Blot Stain (S11791, Thermo Fisher Scientific) according to manufacturer’s instructions. SYPRO Ruby staining was imaged using the Epi Blue setting of the c300 Gel Imaging System.