SUN1,2 (nuclear envelope protein) (Arabidopsis thaliana)
AS15 2856 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana
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|Recommended dilution||1 : 1000 (WB)|
|Expected | apparent MW||
51.5 kDa (SUN1); 49.9 kDa (SUN2)
|Confirmed reactivity||Arabidopsis thaliana|
|Predicted reactivity||Arabidopsis thaliana ,for other species use product AS15 2857|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
Protocol for isolation of cytosolic and nuclear fractions can be found here.
|Selected references||To be added when available, antibody released in June 2016.|
Chloroplast fraction was obtained using Chloroplast Isolation Kit (CPISO-1KT, Sigma-Aldrich). 1. Supernatant proteins after DNA digestion, 2. Unquantified, 10 µl of pellet after DNA digestion. Signal of SMT1 (Sterol methyltransferase 1, marker antibody of integral ER membrane) was not detected. Nuclei fraction was isolated from 1 g fresh weight of 3-4 old day cell suspension culture of Arabidopsis thaliana Ler according to protocol published by Xu and Copeland (2012). Nuclei fraction was stored in NSB buffer (with added ROCHE and PMSF inhibitors) in 80 °C. Residue of nuclei was centrifuged and pellet was re-suspended in 100 µl of modified RIPA buffer with added protease inhibitors and incubated 1h on ice. Then 20 µl of DNase I was added into each sample and all samples were incubated on ice for 45 min. After that 45 min another 20 µl of DNase I was added and incubated under the same conditions. Samples were twice centrifuged 1) 15 000 × g for 15 min and 2) 30 000 × g for 30 min. Pellets from the first step of centrifugation was stored. Proteins in second supernatant were precipitated with ice-cold acetone over night and twice washed. Protein pellet was dissolved in buffer contained 6 M Urea, 2 M Thiourea and 25 mM Tris, pH = 7,5. Proteins were quantified by using Bradford method. Totally 3 µg of protein was loaded on gel. Pellet after DNase treatment was dissolved in 20 µl of LSB buffer. Samples were boiled on 95°C for 10 min before loading on 4 / 12% polyacrylamide gel. Electrophoretic conditions: 90 V during whole separation (2 h); Transfer conditions: 90 mA, 30 V over night, nitrocellulose membrane; Blocking: 1 h, 5% low fat milk; Primary antibody incubation: 1 h at a dilution of 1: 1000; Washes: 3 × 5 min.; Secondary antibody: 1 h at a dilution of 1: 5000; washes: 3 × 10 min. ELC substrate was loaded on membrane and signals were detected with ChemiDoc system (Bio-Rad).
Courtesy of Dr. Martin Kubeš, Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Chemical Biology and Genetics, Olomouc, Czech Republic and Vladimír Skalický, Laboratory of Growth Regulators, Faculty of Science of Palacký University and Institute of Experimental Botany ASCR, Olomouc, Czech republic
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