Liteablot turbo extra sensitive chemiluminescent substrate

Total protein from shoot tissues of pUBQ10:RFP-ANAC017-GFP plants was extracted in a buffer containing 50 mM Tris-HCl pH 7.6, 1 mM EDTA, 100 mM NaCl, 2% SDS and 0.05% Tween-20.
Fifty µg proteins from the extracts, quantified with the Pierce BCA Protein Assay Kit (Thermo-Fisher Scientific), were separated by SDS-PAGE on 10% polyacrylamide Bis-Tris NuPAGE midigels (Thermo-Fisher Scientific) and transferred onto a polyvinylidene difluoride membrane by means of the Trans-Blot Turbo System (Bio-Rad). A monoclonal anti-GFP antibody (Roche, cat. no. 11814460001) was used at 1:3000 dilution and combined with a 1:20000 rabbit anti-mouse secondary antibody (Agrisera, cat. no. AS09627) to detect the Δ533ANAC017:GFP protein fragment. A rat monoclonal anti-RFP antibody (Chromotek, cat. no. 5F8) was used at 1:1000 dilution and combined with a 1:5000 donkey anti-rat secondary antibody (Agrisera, cat. no. AS10947) to detect the RFP:ANAC017Δ24 protein fragment. All antibodies were diluted in 4% milk in TBS-T. Blots were incubated with the LiteAblot Turbo Extra Sensitive Chemiluminescent Substrate (EuroClone) and imaged with UVP VisionWorks LS (Ultra-Violet Products). Equal loading of total protein samples was checked by Amido-black staining of the membrane (Mithran et al., 2014) .