Immobilon p pvdf

For protein separation and detection, the BioRad system, and RunBlue precast gradient gels (expedeon) were used. Prior to loading on the gel, samples were diluted in Laemmli buffer (Laemmli, 1970 (link)) containing 100 mM DTT and heated to 98°C for 10 min. Samples were briefly centrifuged and loaded using a Hamilton syringe. SDS-PAGE was performed at a constant voltage of 150V for 50 min. For Western Blot, Immobilon-P PVDF or nitrocellulose membranes were used in a BioRad system (100V for 90 min while keeping the system cool). Subsequently, membranes were blocked in 5% milk in TBST for 1h and incubated in primary antibody diluted 1:1000 (see KRT for manufacturer and origin and antibody dilutions used) overnight. Membranes were washed 3 times for 5 min in TBST and subsequently incubated in secondary antibody conjugated to HRP (see KRT) for 1h in 5% milk in TBST. After washing 3 times for 5 min, exposure using SuperSignalTM West Pico Plus chemiluminescent substrate (Thermo scientific) or Supersignal West Femto Max Sensitivity ECL onto Lucent Blue X-Ray films (advansta) or Kodak film in the dark was used to detect protein bands. Biochemical fractionation of EGFP-PipB and EGFP-PipB (Δ270–291) was performed as previously described (Lau et al., 2019 ).