DHAR2 | Dehydroascorbate Reductase 2
AS11 1747 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana
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1 : 5000 with standard ECL (WB)
|Expected | apparent MW||
23.6 | 23.4 kDa
|Predicted reactivity||dicots including: Ricinus communis, trees: Populus trichocarpa
|Not reactive in||
no confirmed exceptions from predicted reactivity are currently known
to be added when available
Grefen et al. (2009). The determination of protein-protein interactions by the mating-based split-ubiquitin system (mbSUS). Methods Mol Biol 479:217-233.
1cm2 of a leaf from Arabidopsis thaliana Col-0 (1) and or t-DNA insertion lines dhar1-1 (2), dhar1-2 (3), dhar1-3 (4), dhar2-1 (5), dhar2-2 (6), dhar1-3 EOS-DHAR1 (7), was extracted using 200µl Lyse&Load-Buffer (Grefen et al. 2009). 10 µl were separated on a 15% SDS-PAGE and blotted 1h to PVDF (using Bjerrum Buffer in a semidry blot). Blots were blocked with 5% Milk in 1xTBS-Tween20 (1%) for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:5000 (in 5% Milk 1xTBS-Tween20 (1%) + 0.01 % NaN3) ON at 4°C with agitation. The antibody solution was decanted and the blot was washed 3 times for 10 minutes with 1x TBS-Tween20 at RT with agitation. Blot was incubated in secondary antibody BioRad anti-rabbit IgG AP-conjugate (#170-6518) diluted to 1:2000 in 5% Milk 1xTBS-Tween20 (1%) + 0.01 % NaN3 for 1h at RT with agitation. The blot was washed as above, equilibrated in staining buffer (100mM Tris-HCl, 100mM NaCl, 5mM MgCl2, see Grefen et al. 2009) and developed for 5-15 min. with staining solution (Nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indoylphosphate-p-toluidin (BCIP) in staining buffer).
Courtesy Dr. Chrisopher Grefen, UK
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