LOX | Lipoxygenase (Affinity purified)
AS06 128A | Clonality: Polyclonal | Host: Rabbit | Reactivity: A.thaliana, G. max, S. tuberosum
|Recommended dilution||1 : 5000 (WB)|
|Expected | apparent MW||
54 (subunit), 108 (native enzyme)
|Confirmed reactivity||Arabidopsis thaliana, Glycine max, Solanum tuberosum
Cucumis sativus, Lathyrus undulatus, Malus x domestica
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
|Selected references||to be added when available, antibody released in December 2014|
10 µg of total protein from Arabidopsis thaliana (1), native LOX protein from Glycine max (2,3) total protein from all the samples were extracted with Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70 C for 5 min and keep on ice before loading. Protein samples were separated on NuPAGE 4-12% Tris-Bis gel (Invitrogen) LDS-PAGE and blotted for 1h to 1.5h on PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent (GE RPN 2125; Healthcare) or 5% non-fat milk dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 5 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602) diluted to 1:25 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with ECL detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
Proteins were extracted from tuber flesh of Solanum tuberosum cv. Russet Burbank with 0.1 M Tris HCl (pH=8.0), 5% sucrose (m/v), 2% (m/v) SDS, protease inhibitors (PMSF 1mM). Samples were heated 95°C 5 min, and 10 μg of total protein was resolved in 12% SDS PAGE and blotted to PVDF membrane for 1h-1.5h using tank transfer. Blots were blocked with a skimmed milk 4% (m/v) in T-TBS (1.5h) at RT with agitation. Primary antibodies (AS06 128A) were applied overnight +4°C in dilution 1:5000 with agitation. After washing with T-TBS 2 times, membrane was incubated with secondary antibodies (Goat Anti-Rabbit HRP conjugated) 1:10 000 for 1 hour at RT. Blot was washed as above and developed with ECL (Clarity Western ECL Substrate, BioRad, 170-5060) for 5 – 10 minutes. Exposure time – 7.000 seconds.
Courtesy of Msc. Iauhenia Isayenka, Laboratory of Nathalie Beaudoin, University of Sherbrooke, Canada
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