BIK1 | Botrytis-induced kinase 1
AS16 4030 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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1 : 3000 (WB)
|Expected | apparent MW||44 kDa|
|Confirmed reactivity||Arabidopsis thaliana
|Predicted reactivity||Noccaea caerulescens|
|Not reactive in||
To be added when available, antibody released in November 2017.
Total protein from 7-day old Arabidopsis thaliana seedlings Col-0 (wild-type) and bik1 (null mutant) (Lu et al, 2009) were extracted with 50mM HEPES-KOH buffer containing 250 mM sucrose, 5% glycerol, 50 mM NaPP, 1 mM NaMo, 25 mM NaF, 10mM EDTA, 0.5% PVP, 3mM DTT, 1mM PMSF, 10uM Leupeptin & 10nM Calyculin, and then fractionated by ultracentrifugation at 100,000 x gravity for 30 min at 4°C into soluble (S100) and microsomal (P100) proteins as described in LaMontagne et al. (2016). 30 µg proteins of Total, S100, and P100 fractions were denatured at 65°C for 5 min, separated on a 10 % SDS-PAGE and blotted 1h to nitrocellulose using tank transfer. Blots were blocked with 1x PBS (from Fisher Scientific BP665-1) + 0.1 %Tween 20 (PBS-T) + 5% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 overnight at 4°C with agitation in 1x PBS-T + 5% milk. The antibody solution was decanted, and the blot was rinsed briefly once, then washed three times for 7 min in 1x PBS-T at RT with agitation. Blot was incubated with secondary antibody (Goat anti Rabbit IgG (H&L) –HRP conjugated from Agrisera (AS09 602) diluted to 1:10 000 in 1x PBS-T + 5% milk for 2 hr at RT with agitation. The blot was washed as above and developed for 5 min with Amersham ECL (RPN2106). Exposure time was 6 min.
Courtesy of Gayani Ekanayake & Dr. Antje Heese Division of Biochemistry, Interdisciplinary Plant Group (IPG) - University of Missouri; Columbia, MO, USA
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