Trans blot pvdf membranes

1 × E6 cells/condition were collected and lysed in lysis buffer (50  mM Tris-HCl [pH 7.4], 75  mM NaCl, 1  mM EDTA, 1  mM NaF, and 0.5% NP-40) with a freshly added protease inhibitor cocktail and sodium vanadate and subjected to nine cycles (30 s ON–10 s OFF) of ultrasonication (Bioruptor Plus; Diagenode). The sonicated samples are then spun down, and the supernatant is collected for protein estimation using the microBCA kit (Pierce). 10 µg of protein is then mixed with 1× sample buffer (10% sucrose, 0.1% bromophenol blue, 5  mM EDTA [pH 8.0], 200  mM Tris [pH 8.8]), and boiled at 95°C for 10 min. The lysates are then run on either self-made 10–15% SDS-PAGE gel or on pre-casted Invitrogen NuPAGE 4 bis 12 %, Bis-Tris, 1.0–1.5 mm, Mini-Protein-Gel, followed by blotting with Trans-blot PVDF membranes (Bio-Rad) for 15 min, blocked in 5% BSA in TBS-T for 1 hr before probing with the primary antibodies overnight at 4°C. Secondary antibodies conjugated to HRP were probed for 1–1.5 hr at RT the next day following 3× intensive TBST washing of the unbound primary antibodies. Enhanced chemiluminescence (ECL)-based detection using the WesternBright Sirius Chemiluminescent Detection Kit (Advansta) was performed. Densitometric quantification was performed manually using Fiji (gel analysis tool).