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BRI1 | Brassinosteroid insensitive 1

AS12 1859 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana

BRI1 | Brassinosteroid insensitive 1 in the group Plant/Algal Antibodies / Hormones / Brassinosteroids at Agrisera AB (Antibodies for research) (AS12 1859)

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Background BRI1 (Protein BRASSINOSTEROID INSENSITIVE 1) is a receptor which binds brassinolide and has a dual specificity kinase activity acting on both serine/threonine- and tyrosine-containing substrates. Involved in a signaling cascade including expression of light- and stress-regulated genes, promotion of cell elongation, normal leaf and chloroplast senescence, and flowering. Alternative names:BRI1, BRASSINOSTEROID INSENSITIVE 1, CBB2, CABBAGE 2, DWF2, DWARF 2, BIN1, BR INSENSITIVE 1, ATBRI1, Brassinosteroid LRR receptor kinase
Immunogen KLH-conjugated synthetic peptide derived from Arabidopsis thaliana BRI1 protein, Uniprot: O22476, TAIR: AT4G39400
Host Rabbit
Clonality Polyclonal
Clone
Purity Affinity purified serum in PBS, pH 7.4
Format Lyophilized in PBS pH 7.4.
Quantity 50 µg
Reconstitution For reconstitution add 50 µl of sterile water.
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles.Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications Immunoprecipitation (IP), Western blot (WB)
Related products

AS12 1858 | anti-BAK1 | Brassinosteroid insensitive 1-associated receptor kinase 1, rabbit antibodies
AS16 3203 | Anti-BIN2 | brassinosteroid insensitive 2 antibody
AS16 3204 | Anti-SOBIR1 | supressor of BIR1 antibody

Plant protein extraction buffer

Secondary antibodies

Additional information
application information
Recommended dilution 1 : 5000 (WB)
Expected | apparent MW

above 130 kDa (due to N-glycosylation)

Confirmed reactivity Arabidopsis thaliana
Predicted reactivity Brassica rapa
Not reactive in

Hordeum vulgare, Oryza sativa

Additional information

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.
Selected references

to be added when available, antibody released in July 2014.


Application example

western blot detection using anti-BRI1 antibody

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 Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Exposure time was 3 minutes.

Courtesy of Dr. Elena Petusching, Georg-August-University Goettingen, Germany

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