CPT6 | cis-prenyltransferase 6
AS14 2768 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Microsomal (pellet P) and cytosolic (supernatant S) fractions from Arabidopsis thaliana roots were obtained by homogenization in homogenization buffer (50 mM Tris, pH 7.5, 5 mM MgCl2, 10 μM ZnCl2, 2 mM DTT, 100 mM NaCl, 250 mM sacharose) containing protease (Complete Mini, Roche) and phosphatase (PhosSTOP, Roche) inhibitor cocktails and centrifugation at 200,000 ×g for 1.5 h 25 µg of protein were separated on 12 % SDS-PAGE using wet transfer and blotted 1h to ECL nitrocellulose membrane. Blots were blocked with 4% non-fat milk in PBS-T (0.1% Tween-20 in 1× PBS) for 45 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody 50 µg per 1 ml incubation mixture overnight at 4 °C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed once for 15 min and 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit Gig horse radish peroxidase conjugated, from) diluted to 1:5 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer's instructions. Exposure time was 2 minutes. S1 and S2 cytosolic (supernatant) and P1 and P2 microsomal (pellet) fractions were obtained from two independent experiments. Courtesy Dr. Liliana Surmacz, PAN Poland
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