SGS3 | Protein suppressor of gene silencing 3
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250 µg of grounded tissue from 2 weeks old Arabidopsis thaliana flowers from wt Col-0 and sgs3 mutant was incubated with 1 mL lysis buffer (50 mM HCl pH=7.5, 150 mM NaCl, 10% glycerol, 5mM MgCl2, 0,1% Nonidet P40, Roche protease inhibitors, 1 mM TCEP) and denatured with Laemmli buffer (2% SDS, 10% glycerol, 0.125 M Tris-HCl pH=6.8, 1% DTT) at 85°C for 7 min. The samples were separated on 12% SDS-PAGE and blotted for 60 min to nitrocelulose membrane using wet transfer. Blots were blocked with 5% milk powder in PBST for 180 min in a cold room with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for O/N in a cold room with agitation. The antibody solution was decanted and the blot was rinsed briefly once, then washed 5 times in TBST/PBST buffer at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602, Agrisera) diluted to 1:10 000 in for 60 min at RT with agitation. The blot was washed as above and developed for 1 min with (ECL DIY mix (100 mM Tris pH8.8, 2 mM 4IBPA (SIGMA), 1.25 mM Lumminol (SIGMA)) using X-ray film. Exposure time was 60 seconds.
Courtesy of Maria Louisa Vigh, Department of Computational and RNA Biology, University of Copenhagen, Denmark
Protein supressor of gene silencing 3 (SGS3) is a protein which is necessary for post-transcriptional gene silencing and virus resistance.
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