Criterion tgx stain free pre cast gels

Samples (40 μl) were heated at 65°C for 30 min before separation by SDS–PAGE using 12% Criterion TGX Stain-Free pre-cast gels (Bio-Rad) and a Tris/glycine/SDS running buffer (Bio-Rad) at 240 V for 35 min. Proteins were transferred to a 0.2 μm PVDF (polyvinylidene fluoride) membrane using the Trans-Blot Turbo transfer system (Bio-Rad) according to the manufacturer’s protocol. The membrane was blocked for 20 min at room temperature with 5% (w/v) skimmed milk in PBS-T buffer and incubated for 1 h with the C-terminal sequence-specific VpVAN primary antibody at a 1 : 300 dilution in 2% (w/v) skimmed milk in PBS-T. The blot was washed with PBS-T buffer and incubated with a secondary swine anti-rabbit horseradish peroxidase-conjugated antibody (Dako) using a 1 : 5,000 dilution in PBS-T for 1 h at room temperature. The membrane was washed again with PBS-T buffer and the secondary antibody detected using SuperSignal West Dura Chemiluminescent Substrate (Pierce) and developed for 1–10 min with a ChemiDoc MP Imaging System (Bio-Rad). A specific antibody to the PSI-D (Haldrup et al. 2003 (link)) in a 1 : 10,000 dilution was used as a reference in quality tests of the isolated chloroplasts.