Anti rat hrp

Expression of MRP4 was monitored by Western blot. The total protein concentration
of membranes was measured using a bicinchoninic acid (BCA) assay kit (Pierce,
Thermo Scientific, Waltham, MA). Specified amounts (µg) of total protein were
loaded on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, and
blocked with 5% (w/v) bovine serum albumin (BSA) in TBS-T (20 mM Tris, pH 7.5,
150 mM NaCl, 0.01% [v/v] Tween-20). Blots were probed with either a mouse
anti-his antibody (R&D Systems, Abingdon, UK) at a dilution of 1:500 or a
rat anti-MRP4 antibody (M4I-10, Enzo, Exeter, UK) at 1:100, followed by
anti-mouse HRP (Cell Signaling, London, UK, 1:3000) or anti-rat HRP (Sigma,
Gillingham, UK, 1:3000). All were visualized using chemiluminescence (Pierce)
and a C-Digit Western blot scanner (Licor, Cambridge, UK).

Total protein extracts were obtained from 3- to 4-week-old leaves ground to fine powder in liquid nitrogen and boiled at 95°C for 5 min in Sample buffer [65.8 mM Tris–HCl Buffer, pH 6.8, 25% (w/v) glycerol, 2% SDS, 0.01% (w/v) bromophenol blue, and 5% (w/v) 2-mercaptoethanol]. Proteins were separated on 8% SDS-PAGE gels and electroblotted onto PVDF membranes (Biorad Mini Trans-Blot^® Electrophoretic Transfer Cell). Membranes were blocked in 5% (w/v) BSA in PBS overnight at 4°C. Anti-HA antibody (Roche) was diluted in 0.5% (w/v) BSA in PBS–Tween 0.1% (w/v) solution to 1:1000 and incubated for 1 h at room temperature. Secondary anti-rat-HRP (Sigma) was diluted 1:2000 and incubated 1 h at room temperature as well. Bands were visualized using chemiluminescent substrate SuperSignal West Femto Kit (Thermo) before exposure to film (Biomax light film Sigma–Aldrich). Membranes were stained with Coomassie Blue to check for equal loading (Welinder and Ekblad, 2011 (link)). Samples from four plants were pooled, experiments were performed twice.