Anti-SYP121 | Syntaxin 121

Samples:
Marker: Precision Plus Dual Color Standard (Biorad)
1-20 µg of Arabidopsis thaliana total protein extract
2-20 µg of Arabidopsis thaliana cytosolic fraction
3-20 µg of Arabidopsis thaliana microsomal fraction
4-20 µg of Arabidopsis thaliana plasma membrane fraction
5-20 µg of Arabidopsis thaliana other membranes fraction

Arabidopsis thaliana plants were grown for 13 days at 22°C with a time regime of 16hday/8h night. Plants were grown on 1/2MS agar plates, pH=5.8. Whole plants were used for the experiment.

Samples were extracted with 50 mM HEPES pH 7,5, 400 mM sucrose, 100 mM KCl, 100 mM MgCl2, Complete Protease Inhibitor Cocktail; 3-8 resuspend with 5 mM phosphate buffer pH 7,8 and denaturated with Laemmli sample buffer (Biorad) with XT reducing agent (Biorad) at 65 °C (1-5) or 70 °C (6-8) for 5 min. Samples were separated on 4-20% Mini-PROTEAN TGX stain free precast gel and blotted 7 min to PVDF (pore size of 0,2 µm) using Turbo Blot. Blot was blocked with 5% milk in TBS-T for 1h/RT with agitation. Blot was incubated with antiSYP121 (AS 22 4827) at a dilution 1:1000 ON/4°C in TBS-T. The antibody solution was decanted and the blot was washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Goat anti-Rabbit IgG HRP conjugated (Agrisera AS09602-trial) diluted to 1:25000 in 5% milk in TBS-T for 1h/RT with agitation. The blot was washed as above and developed for 5 min with Agrisera ECLBright. Exposure time was 2 seconds.

Courtesy of Dr. Roman Hudeček, Integrative Structural Biology, IEB CAS CZ, Czech Republic