Tgx fastcast acrylamide solution

For optimal condition identification, we prepared soluble and insoluble fractions as described. The samples were normalized to OD₆₀₀ = 25 before harvesting. Cell pellets were resuspended with 300 µL phosphate-buffered saline (PBS) before sonicating and centrifuging at 10,000 xg for 10 min. The supernatant was collected as the soluble fraction of the whole-cell lysate. The pellet part was washed 2 times with 500 µL TE buffer (50 mM Tris–HCl pH 8.0 and 1 mM EDTA), resuspended with PBS containing 2% w/v SDS and boiled at 100 °C for 5 min then centrifuged at 10,000 xg for 10 min. The supernatant was collected as the insoluble fraction. Samples were mixed with 6 × gel loading dye (Bio-Rad) containing 10% v/v β-mercaptoethanol, then boiled at 100 °C for 5 min. Next, 40 µL of samples were loaded onto 10% polyacrylamide gels that were prepared according to the manufacturer’s protocol (TGX FastCast Acrylamide Solutions; Bio-Rad). Protein staining was performed using InstantBlue® Coomassie Protein Stain (Abcam). Gels were soaked overnight and were washed the next day with washing buffer (10% v/v acetic acid mixed with 10% v/v methanol). Gel images were captured by Gel Doc EZ Gel Documentation System (Bio-Rad). We ensured purified protein samples by quantifying all samples using the Bradford assay, and 0.5 µg of each fraction was loaded per well.

Protein samples were suspended in sample buffer (50 mM Tris pH 6.8, 8% glycerol, 2% SDS, 20% β-mercaptoethanol, containing bromophenol blue), heated to 95 °C for 3 min and separated by electrophoresis on 10 or 12% self-made Tris-glycine poly-acrylamide (Serva) or TGX FastCast acrylamide solution (Bio-Rad) gels using Laemmli (25 mM Tris, 192 mM glycine, 0.1% SDS) running buffer at 30 mA/gel.

Cells were washed by adding cold PBS, and then scraped and lysed in SB 1X (45 mM Tris-HCl, pH 7.6, 10% Glycerol, 2% SDS, 1.5% 2-mercaptoethanol, 0.001% bromophenol blue) or RIPA (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X100, 0.5% DOCA, 0.1% SDS) buffer supplemented with 1X protease inhibitors (P8340, Sigma-Aldrich). Protein lysates were sonicated for 5 s and boiled to 95 °C for 5 min before being loaded on a 4–12% TGX Gel (TGX FastCast Acrylamide Solution, Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer’s instructions. Proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (162-0177, Bio-Rad) using the Trans-Blot Turbo System (Bio-Rad). The membrane was blocked with 5% nonfat milk or BSA (bovine serum albumin) in Tris-buffered saline with Tween 20 (20 mM Tris-HCl, pH 7.6, 137 mM NaCl 0.1% Tween 20) and then incubated with primary antibodies overnight at 4 °C. Membranes were washed 3 times with TBS-Tween 20 0.1%, and then incubated with secondary anti-rabbit or anti-mouse HRP-conjugated antibody according to the manufacturer’s instructions (BI2407, BI2413C, Paris, France). The membrane was then washed 3 times with TBS-Tween 20 0.1% and incubated with Clarity Western Cl substrate (1705051, Bio-Rad), and chemiluminescence was monitored using a Bio-Rad ChemiDoc XRS+.