Cyt f | Cytochrome f protein (PetA) of thylakoid Cyt b6/f-complex (higher plants)
AS08 306 | Clonality: Polyclonal | Host: Rabbit | reactiviy: A. thaliana, E. crus-galli, H. vulgare, N. tabacum, P. miliaceum, P. abies, P. sativum, T. elongatus, S. lycopersicum, Z. mays
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maize cytochrome f purified from chloroplasts, including a final gel purification on a denaturing gel. protein used to elicit this antibody is conserved in Arabidopsis thaliana cyt f UniProt: P56771, TAIR: AtCg00540
1.0 µg of chlorophyll from pea (C3 plant) and from mesophyll (M) and bundle sheath (BS) thylakoids of various treatments of Zea mays, Echinochloa crus-galli, Panicum miliaceum (C4 plants) extracted with 0.4 M sorbitol, 50 mM Hepes NaOH, pH 7.8, 10 mM NaCl, 5 mM MgCl2 and 2 mM EDTA. Samples were denatured with Laemmli buffer at 75 0C for 5 min and were separated on 12% SDS-PAGE and blotted 30 min to PVDF using wet transfer. Blot was blocked with 5% milk in TBS for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 3000 overnight at 40C with agitation in 1% milk in TBS-T. The antibody solution was decanted and the blot was washed 4 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602, Lot 1711) diluted to 1:25 000 in 1 % milk in TBS-T for 1h at RT with agitation. The blot was washed 5 times for 5 min in TBS-T and 2 times for 5 min in TBS, and developed for 1 min with 1.25 mM luminol, 0.198 mM coumaric acid and 0.009% H2O2 in 0.1 M Tris- HCl, pH 8.5. Exposure time in ChemiDoc System was 125 seconds.
Courtesy of Dr. Wioleta Wasilewska-Dębowska, Warsaw University, Poland
Thylakoid membranes (10 µg of total chlorophyll) extracted freshly from Hordeum vulgare leaves with 100 mM HEPES-KOH (pH 7.5), 0.3 M sorbitol, 2 mM EDTA, and 1mM MgCl2 and denatured with a Laemmli buffer at 80°C for 5 min were separated on 12% SDS-PAGE and blotted 1 h to nitrocellulose (pore size of 0.2 um), using semi-dry transfer. Blot was blocked with 4% milk for 2 h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:3000 (PC and PetA, simultaneous western blot detection for both antibodies at the same time) for 1 h/RT with agitation in PBS-T. The antibody solution was decanted and the blot was rinsed briefly, then washed 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:25000 in for 1 h/RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescent detection reagent according to manufactures recommendations. Exposure time was 30 seconds. Simultaneous western blot detection can be applied if MW of detected proteins differs in min. 20 kDa.
Courtesy Dr. Anja Liszkay, CNRS, France
Multi-subunit complex of cytb6/f is a crucial component for the photosynthetic electron transport chain of higher plants, green algae and cyanobacteria. This complex is catalyzing oxidation of quinols and the reduction the reduction of plastocyanin. This reaction allows to establish the proton force required for the ATP synthesis. Four major subunits build the complex: the petA gene product corresponding to a c-type cytochrome (cytf), the petB gene product corresponding to a b-type/c’-type cytochrome with three haems (cyt b6), the petD gene product (subunit IV, or suIV), and the petC gene product, corresponding to the Rieske/Iron/sulfur protein.
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