G4 | Chlorophyll synthase (chloroplastic)
AS14 2793 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Hordeum vulgare
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41.8 kDa (further processed into a mature form)
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Barley seeds were grown for 4.5 days in the dark before they were harvested and intact plastids were isolated in light according to Eichacker et al. 1996. The concentration of plastids was determined by counting in a Thoma counting chamber. The membrane complexes were isolated according to Reisinger et al. 2008 before 3x SB buffer (2% (w/v) SDS, 10% (w/v) sucrose, 0.03% (w/v) bromphenol blue, 66 mM NaCO3, 66 mM dithiothreitol) was added. The proteins were denatured for 2 min at 72 °C. The membrane fractions were loaded onto a 12.5 % SDS-acrylamide gel, giving approximately 2.5x107 plastids per lane alongside with Magic Marc (LC5602, Life technologies). The gel was then run at 175 V with 1xSDS running buffer. The proteins were then transferred onto a nitrocellulose membrane by using semi-wet transfer and Towbin buffer w/10 % methanol. The proteins were transferred at 2 mA/cm2 for 1 hour. The membrane was blocked with TBS-milk, washed 3x5 min in TBS-tween and the primary antibody (Chlorophyll synthase, AS14 2793 from Agrisera) was added at a concentration of 1:1000. The membrane was incubated ON at 4 °C (approximately 18 h) before washing with TBS-tween and incubation for 1h at RT with the secondary antibody diluted 1:12 500 (Agrisera AS09 602). After washing, the membrane was incubated in an ECL1/ECL2 solution (100 mM Tris, 2.5 mM Luminol, 0.4 mM pCoumaracid/100 mM Tris, 0.0183 % Hydrogenperoxide) before exposure to a film for 7 min. The film was then developed and fixed using chemicals from Kodak.
Courtesy of Eli Drange Vee, Staff Engineer, Centre for Organelle Research, University of Stavanger, Norway
G4 (Chlorophyll synthase) is an enzyme which performs estrification of chlorophyllide (a and b), which is the last step of chlorophyll biosynthesis. Alternative names: polyprenyl transferase, protein G4.