RPS6A-P240 | Phosphorylated (Ser240) 40S ribosomal protein S6-1
AS19 4302 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Left panel is developed with anti-RPS6-p240, while right panel anti-RPS6, which serves as a loading control.
1 and 4 - 0.5 µg of Arabidopsis thaliana whole leaf extract from untreated leaf discs
2 and 5 - 0.5 µg of Arabidopsis thaliana whole leaf extract from leaf discs treated with 10 mM glutamine (TOR activator) for 8 hours
3 and 6 - 0.5 µg of Arabidopsis thaliana whole leaf extract from leaf discs treated with 2 µM AZD-8055 (TOR inhibitor) for 8 hours
0.5 µg/well of total protein extracted freshly from mature Arabidopsis thaliana leaves with 50 mM HEPES pH 7.5, 5mM NaF, 2.5 mM NaPPi, 25 mM B-phosphoglycerol, Roche Complete inhibitor tablet (1x), 2% PVPP, 2 mM PMSF) and denatured with 2x SDS sample buffer (80 mM tris pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT, bromophenol blue) at 99°C for 3 min. were separated on AnyKd (BioRAD) gradient % SDS-PAGE and blotted 1h to PVDF (pore size of 0.45 um) using semi-dry transfer. Blot was blocked with 2% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 in TBS-T + 2% milk powder ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 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 + 2 % milk powder for 1h/RT with agitation. The blot was washed as above and developed for 5 min with Agrisera ECLBright with GE Amersham Imager 600. Exposure time was ~60.seconds.
Courtesy of Dr. Brendan O'Leary, University of Western Australia, School of Molecular Sciences, ARC Centre for Plant Energy Biology, Australia
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