Extra thick blot paper

Test strips were manufactured according to Posthuma-Trumpie, Korf & Van Amerongen (2008) (link). Briefly, a Linomat V (Camag, Muttenz, Switzerland) was used to dispense antibodies onto a nitrocellulose membrane of 2.5 cm wide. For the control line, goat anti-mouse IgG (M5899; Sigma, St. Louis, MO, USA) was dispensed at a dose of 2.0 µg cm⁻¹ at the position two cm away from the dipping point. For the test line, capture antibody (10-2698 from Fitzgerald or HM026 from EastCoast Bio) was dispensed at a dose of 0.5–2.0 µg cm⁻¹ at the position 1.5 cm away from the dipping point. The membrane was then dried for 2 h at 37 °C. An absorption pad (Extra Thick Blot Paper, BIO-RAD, Hercules, CA, USA) was applied to the dried membranes, which were then cut to a width of four mm by an Autokun cutter (Hangzhou Autokun Technology, Hangzhou, China). Finally, test strips were sealed in aluminum packages with a desiccation pad and stored at 4 °C until use. Three nitrocellulose membrane types were tested, namely CNPC-SS12, 10 µm with wicking time of 140 ± 28 s/40 mm (MDI Technologies, Ambala Cantt, India), UniSart^® CN140 with wicking time of 95–155 s/40 mm, and UniSart^® CN 95 with wicking time of 65–115 s/ 40 mm (Sartorius, Goettingen, Germany).

Transfer apparatus; Bio-Rad Trans-Blot SD cell

Blotting paper; Bio-Rad extra thick blot paper, Cat. no. 1703965

Acetic acid; Thermo Fisher Scientific, Cat. no. A38C-212

Ethanol: 200 proof (100%); Thermo Fisher Scientific Cat. no. 04-355-450

Protocol and reagents adapted from Sancak et al (2013) (link). After electrophoresis was complete, the gels were transferred to Bio-Rad Mini-size 0.22 μm PVDF membranes in Invitrogen Novex Tris–glycine transfer buffer at 0.18 A for 20 min, using a Bio-Rad TransBlot SD Semi-Dry Transfer Cell and extra thick blotting paper. Membranes were incubated in 8% acetic acid while shaking for 15 min to fix the proteins. The membranes were rinsed with dH₂O for 5 min, and then air-dried. Once dry, the membranes were rehydrated with ethanol. The membranes were then blocked with 5% milk in TBST (wt/vol) and immunoblotted using FLAG antibody as described above. Finally, the same membranes were probed for ATP5A (a mouse antibody) as described above, as a loading control.