BAK1 | Brassinosteroid insensitive 1-associated receptor kinase 1
AS12 1858 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana, Solanum lycopersicum
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|Recommended dilution||1: 5000 (WB), 2 µl/50 µl of Protein G agarose|
|Expected | apparent MW||
68 | 70 kDa
|Confirmed reactivity||Arabidopsis thaliana, Solanum lycopersicum
|Predicted reactivity||Thelungiella halophila - for other species, please inquire.|
|Not reactive in||
Hordeum vulgare, Nicotiana benthamiana, Oryza sativa
|Additional information||Extra Information on CE extraction buffer: CE buffer does not need to be made freshly everytime. Aliquots can be kept at -20°C. Na2MoO4 and NaF are phosphatase inhibitors, included to prevent lose phosphorylation form our protein of interest during extraction. EDTA chelates metal ions and thus inhibits many enzymes which need metal ions as co-factors and inhibits the action of proteases. Protease inhibitor coctail is Sigma product number P 9599 which is used in dilution 1: 100.|
|Selected references||Bundy et al. (2016). A mutation in the catalytic subunit of the glycosylphosphatidyl inositol transamidase disrupts growth, fertility and stomata forma formation in Arabidopsis. Plant Physiology. DOI:10.1104/pp.16.00339.
Tateda et al. (2014). Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling.Plant Cell. 2014 Oct 14. pii: tpc.114.131938.
Plant material: Col-0 (wild type) bak1-4 (knock-out mutant for BAK1, does not show any full-length BAK1 transcript, Kemmerling et al., 2007). Method: Two-week-old seedlings and leaf material of 5-week-old plants (grown under short day conditions, 8h light) were collected and frozen in liquid nitrogen. Approximately 100mg of plant material were extracted in 0.2 ml of homogenization buffer (250 mM sucrose, 50 mM HEPES-KOH pH 7.5, 0.5% Triton X-100, 5% glycerol, 50 mM Na4P2O7, 1 mM Na2MoO4, 25 mM NaF, 2 mM DTT, Sigma plant protease inhibitor cocktail) with a glass pistil and a small amount of sand in an 1.5ml Eppendorf tube. Another 0.8 ml of buffer were added and the extract was mixed thoroughly. Debris was pelleted by centrifugation in a table-top microcentrifuge at 13 000 rpm. The supernatant was mixed with 4x SDS loading buffer (200 mM TRIS-HCl pH 6.8, 400 mM DTT, 8% SDS, 40% glycerol, 0.1% bromophenol blue) and boiled at 95°C for 5min. 10µl were run on an 10% polyacrylamide gel and blotted onto a 0.45µm PVDF membrane (Carl Roth). The membrane was blocked for 1h in TBS-T (150mM NaCl, 10mM Tris-HCl pH8, 0.05% Tween-20) containing 5% skimmed milk powder. The primary antibody (Agrisera Rabbit anti-BAK1, AS12 1858, 1µg/µl) was diluted 1:5000 in TBS-T containing 5% milk powder and incubated on the membrane overnight at 4°C. Then the membrane was washed 5 times 15min with TBS-T containing 5% milk powder. The secondary antibody (Agrisera Goat anti-rabbit IgG (H&L) HRP conjugate, AS09 602, 0.91 µg/µl) was diluted 1:5000 in the same solution and incubated on the membrane at room temperature for 2h. The membrane was then washed 5 times 15min with TBS-T (no milk powder) and the blot was developed using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Short exposure: 1 minute. Long exposure: 10 minutes.
Courtesy of Dr. Elena Petusching, Georg-August-University Goettingen, Germany
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