ASY1 | Asynapsis 1

0.25 µg/well of total overexpressed Hordeum vulgare ASY1 protein extracted the previous day from Rosetta 2 pLysS cells with BugBuster master mix and Complete EDTA free protease inhibitor, captured with NiNTA, digested at 4°C overnight with ProTEV protease to remove the affinity tag, and run through a second round of NiNTA capture to remove the cleaved affinity tag. Each lane in the gel also includes His-tagged ASY1 protein. Denaturation was performed with LDS buffer, DTT, and Urea at 90°C for 10 min. Samples were separated on 4-12% NuPAGE Bis-Tris SDS-PAGE at 200v for 1h in MOPS buffer with 0.5 ml NuPAGE antioxidant then transferred to PVDF (pore size of 45 µm) at 11 v overnight in NuPAGE transfer buffer with 0.5 ml NUPAGE antioxidant. The blot was fixed in methanol, washed in SDW then blocked with 5 % milk 1h/RT with agitation. The blot was incubated in the primary antibody at a dilution of 1: 5000 for 2h/RT with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. The blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:25000 in for 1h/RT with agitation. The blot was washed as above and developed for 3 min with Agrisera ECLSuperBright. The exposure time was 37 seconds.

Courtesy Jamie Orr, The James Hutton Institute, United Kindgdom