SUN1,2 | SUN domain-containing protein 1,2

Rabbit

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Samples:
1 - 15 ug of Arabidopsis thaliana Col-0 callus total protein
2 - 15 ug of Arabidopsis thaliana sun1 mutant callus total protein
3 - 15 ug of Arabidopsis thaliana sun1 sun2 mutant callus total protein
MW markers: PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (ThermoFisherScientific)

15 µg/well of total protein extracted freshly from Arabidopsis thaliana callus with 50 mM Tris pH7.5, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 1xprotease inhibitor cocktail (ThermoFisherScientific) and denatured with Bio-Rad 1x Laemmli sample buffer (BioRad) at 95°C 5 min. Samples were separated on 12% SDS-PAGE  and blotted 15V, 16h to PVDF (pore size of  45 µm), using wet transfer. Blot was blocked with 5% BSA for 1h/RT agitation. Blot was incubated in the primary antibody in 2% BSA in TBS-Tat a dilution of 1: 1 000 for 1h/RT with agitation in 2% BSA with 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. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated AS09 602) diluted to 1:25 000 in TBS-T for 1h/RT with agitation. The blot was washed as above and developed for 2 min with Agrisera ECLBright. Exposure time was 10 seconds.

Courtesy of Wei Wang, Umeå Plant Science Centre, Sweden