BRI1 | Brassinosteroid insensitive 1
AS12 1859 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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above 130 kDa (due to N-glycosylation)
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Hordeum vulgare, Oryza sativa, Solanum lycopersicum
15 µg of total protein from leaf material of 5 week-old plants of Arabidopsis thaliana, were extracted with homogenization buffer (250 mM sucrose, 50 mM HEPES-KOH pH 7.5, 5% glycerol, 0.5% Triton X-100, 50 mM Na4P2O7, 1 mM Na2MoO4, 25 mM NaF, 2 mM DTT, Sigma plant protease inhibitor cocktail). 3 parts of protein extract were mixed with 1 part of standard SDS loading buffer (200 mM TRIS pH=6.8, 400 mM DTT, 8% SDS, 40% glycerol, 0.1% bromophenol blue). Protein denaturation was done at 90°C/5 min. Proteins were separated on a 10 % SDS-PAGE and blotted using BioRad Tank Blot device onto a PVDF membrane at 100 V for 1 h 15 using following blotting buffer: 50 mM TRIS-base, 50 mM boric acid of pH of 8.3. Blots were blocked with TBS-T (150mM NaCl, 10mM Tris-HCl pH8, 0.05% Tween-20) containing 5% skimmed milk powder for 1h at room temperature (RT) with agitation. The blot was incubated in the primary antibody at a dilution of 1: 5 000 in TBS-T with milk powder overnight at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 5 times for 15 min in TBS-T (with milk powder) at RT with agitation. The blot was then incubated in secondary antibody (Agrisera Goat anti-rabbit IgG (H&L) HRP conjugate, AS09 602) diluted to 1:5000 in TBS-T (with milk powder) for 2h at RT with agitation. The blot was washed 5 times for 15 min in TBS-T (without milk powder) and developed using chemiluminescent detection. Exposure time was 3 minutes.
Courtesy of Dr. Elena Petusching, Georg-August-University Goettingen, Germany
Antibody was tested on bri1-1 and bri1-5 mutants. Bri1-1 is a point mutation in the kinase domain that renders the protein non-functional and plants compensate for that by over-accumulating the non-functional receptor. Bri1-5 is a mutant in the extracellular domain and the bri1-5 protein is retained in the ER. The bri1-5 plants contain less protein than the wild type and show an intermediate brassinosteroid deficient phenotype. Also BRI1-5 migrates higher than wild type BRI1 in SDS-PAGE, because it carries ER-type high mannose N-glycans.For IP: 15 µl GFP-trap beads was used for 200 mg plant material to precipitate GFP-tagged protein followed by detection with Co-IPed BRI1 on Western with 1:5000 diluted anti-BRI1 antibody.
Protein extraction has to be done efficiently as this step is crucial, recommended material to buffer ratio: 15 µl/µg or less.
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