HY1 | Heme Oxygenase 1
AS12 2636 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Arabidopsis thaliana wild-type (Col-0) and hy1-100 mutant seedlings were grown on ½ MS agar plates supplemented with 1% agar, without sucrose, for 2 d dark and 3 d in WLc (100 µmol m-2 s-1) at 22°C. 100 mg of cotyledon tissue was collected from 5 d old seedlings and extracted with 500 µL 2x Laemmli buffer without bromophenol blue ( 0.125 M Tris-HCl, pH 6.8; 4 % SDS; 20 % glycerol and 5 % 2-mercaptoethanol ), denatured at 99°C for 5 min using thermomixer (Eppendorf), and centrifuged for 5 min at 13,000 rpm at 4°C. Approximately 100 µg protein from 2 mg of leaf material was loaded per lane (in a 10 µL volume) with appropriate reductions for 0.5x and 0.25x, respectively. Proteins were separated on a 4 % stacking and 12 % resolving SDS-PAGE gel using standard Tris/Glycine running buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS) and blotted for 1 h at 100 V to a 0.22 µm nitrocellulose membrane from Licor. A wet transfer was used, with transfer buffer containing: 20 % methanol, 25 mM Tris, 192 mM glycine. Blots were stained with Ponceau S solution (0.1 % Ponceau S in 5 % acetic acid) and washed briefly with TBS, then blocked with 5% (w/v) milk in TBS for 1 h at room temperature (RT) with agitation. Blots were incubated in the primary antibody at a dilution of 1:2000 overnight at 4°C with agitation in 5 % milk in TBS-T. The antibody solution was removed, the blot rinsed briefly, and washed six times for 5 min in TBS-T at RT with agitation. The blot was incubated in a secondary antibody protected from light (donkey anti-rabbit IgG IRDye800CW from Licor, 925-32213) diluted to 1:20000 in 5 % (w/v) milk in TBS-T for 45 min at RT with agitation. The blot was washed six times for 5 min in TBS-T at RT with agitation and three times for 2 min with TBS to remove residual Tween. For visualization, the blot was excited with the 700 nm and 800 nm channels, with scanning intensity 3 and 5, respectively, using the Odyssey infra-red blot scanner from Licor.
Courtesy of Sylwia Kacprzak and Dr. Matthew J. Terry, University of Southampton, United Kingdom
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