Gradient tgx gels

Samples collected from each purification step were analysed by SDS-PAGE and Western blot. For SDS-PAGE, precast gradient TGX gels (Bio-Rad) were used. Gels were either stained by Instant Blue (Expedeon) or immediately transferred to PVDF membrane (Bio-Rad) at 100V for 1 h. The proteins on the PVDF membrane were probed with two primary antibodies, rabbit anti-Gs C-18 antibody (cat no. sc-383, Santa Cruz) against Gαs subunit and mouse penta-His antibody (cat no. 34660, QIAGEN) against His-tags. The membrane was washed and incubated with secondary antibodies, 680RD goat anti-mouse and 800CW goat anti-rabbit (LI-COR). Bands were imaged using an infrared imaging system (LI-COR Odyssey Imaging System).

Llama serum and EV isolates (an EV pellet derived from 100 μl serum, reconstituted in 100 μl DPBS after isolation and purification) were diluted 1:1 in 2x Laemmli sample buffer, boiled for 5 min at 100 °C and separated by SDS-PAGE on 4–20 % gradient TGX gels (BioRad UK). Approximately 5 μg protein was loaded per lane and transferred to nitrocellulose membranes using semi-dry Western blotting. Blocking of membranes was performed in 5 % BSA in TBS-T for 1 h at room temperature (RT) and incubation with primary antibodies, diluted in TBS-T, was carried out at 4 °C overnight (F95 MABN328, Merck, 1/1000; PAD2 ab50257, Abcam, 1/1000; PAD3 ab50246, 1/1000; PAD4 ab50247, 1/1000; citH3 ab5103, 1/1000; CD63 ab216130, 1/1000; Flot-1 ab41927, 1/2000). The membranes were washed in TBS-T for 3 × 10 min at RT and thereafter incubated in the corresponding secondary antibody (anti-rabbit IgG BioRad or anti-mouse IgM BioRad, diluted 1/4000 in TBS-T) for 1 h at RT. Membranes were washed for 6 × 10 min in TBS-T and visualisation performed using electrochemiluminescence (ECL) and the UVP BioDoc-ITTM System (Thermo Fisher Scientific, UK).