TROL | Thylakoid rhodanese-like protein
AS19 4257 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Pisum sativum, Nicotiana tabacum
TROL protein can be used as a chloroplast dual-localization marker: for the thylakoids (the major portion of the protein) and the chloroplastic inner envelope. Since the envelopes make a very small portion of the total membranes, the envelope form of TROL is detectable only when isolated envelopes are applied.
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Western blot on Arabidopsis thaliana chloroplasts (2 µg of chlorophyll) using anti-TROL antibodies: Chloroplasts were isolated from 5 week old Arabidopsis thaliana by mixing and filtering through 1-layer Miracloth filter and 4-layers of gauze. Chloroplasts were isolated in 330 mM Sorbitol, 20 mM Tris/HCl pH 8.4, 5 mM EDTA, 10 mM Na2CO3, 0.1% BSA and washed twice in the same buffer and pelleted at 1000g for 5 min at 4°C. Chlorophyll concentration was determined and sample corresponding to 2 μg chlorophyll was separated by 12 % SDS-PAGE. After wet Western transfer for 1h15min on nitrocellulose membrane (0.45μm WC), blot was blocked with 5% milk for 3 times for 10 min at RT with agitation. Blot was incubated in the anti-TROL primary antibody at a dilution of 1:2000 at 4°C/ON with agitation, in PBS buffer containing 1%Tween 20 and 5% milk. The antibody solution was decanted and the blot was rinsed briefly, then washed 3 times for 10 min in the same buffer at RT with agitation. Blot was incubated in anti-rabbit IgG horse radish peroxidase conjugate diluted to 1:50 000 in PBS buffer containing 1%Tween 20 and 5% milk for 1h/RT with agitation. The blot was washed 2 times for 10 min in PBS buffer containing 1% Tween 20 and developed using 1 ml 1.13 mM luminol (Alfa Aesar) solution in 0.1M Tris(HCl) pH 8.6, 100 μl 6.7 mM p-hidroxy coumaric acid (Sigma) in DMSO and 0.3 μl 35% H2O2 (Kemika). ECL results was analysed by 3-minute exposure on X-ray films.
Courtesy of Dr. Lea Vojta and Dr. Hrvoje Fulgosi, Laboratory for Molecular Plant Biology and Biotechnology, Division of Molecular Biology, Institute Ruđer Bošković,Croatia
Western blot on TROL-overexpressing Arabidopsis thaliana line using anti-TROL antibodies: TROL overexpression line was constructed by transformation of knock-out Arabidopsis thaliana plants by plasmid vector pH7WG2.0 containing TROL-HA-FLAG construct. Total leaf extract from Arabidopsis thaliana corresponding to 20 mg of wet leaf tissue and isolated chloroplasts corresponding to 25 µg of chlorophyll, were loaded on 15 % SDS-PAGE. Western transfer was performed for 1 hour at 200 mA (wet blot). Membrane was immunodecorated with anti-TROL (1:2000) and anti-HA (1:1000) primary antibodies overnight at 4°C. As secondary antibody, anti-rabbit IgG-HRP conjugate (1:50000) was used for anti-TROL and anti-rat IgG-HRP conjugate (1:30000) for anti-HA. Detection was performed using ECL and exposure on X-ray film.
Courtesy of Dr. Lea Vojta, Laboratory for Molecular Plant Biology and Biotechnology, Division of Molecular Biology, Institute Ruđer Bošković, Croatia
For AgriseraECLBright and Agrisera matching secondary antibodies goat anti-rabbit HRP conjugated, dilution 1: 25 000 1h/RT incubation (AS09 602), anti-TROL antibodies can be used in dilution of 1:3000.
Recommendation: 2 µg chlorophyll/well. With increased protein or chlorophyll load/well background bands may be detected. Check here.
Alternative names: Protein THYLAKOID RHODANESE-LIKE1, Sulfurtransferase 4, AtStr4
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