SUN1 | SUN domain-containing protein 1
AS18 4224 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
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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
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