TROL | Thylakoid rhodanese-lik

TROL (thylakoid rhodanase-like protein) is an integral membrane component associated to the photosynthetic apparatus of higher plants. TROL is involved in the final step of photosynthetic electron transport by binding a key energy-conversion enzyme ferredoxin: NADP+ oxidoreductase (FNR). This interaction enables direct transfer of photosynthetic electrons from iron-sulphur protein ferredoxin at the stromal site of photosystem I to FNR, which then hands over electrons to NADP+. TROL is located in two distinct chloroplast compartments – in the inner envelope of chloroplasts, in its precursor form; and in the thylakoid membranes, where it is processed completely. Alternative names: Protein THYLAKOID RHODANESE-LIKE1, Sulfurtransferase 4, AtStr4
Application example



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 thalianaplants 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