FLS2 | Flagellin-sensitive 2
AS12 1857 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana
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|Recommended dilution||1: 5000 (WB)|
|Expected | apparent MW||
126 kDa (without propeptide)
|Predicted reactivity||Capsella rubella, Glycine max, Hordeum vulgare, Populus trichocarpa, Vitis vinifera
|Not reactive in||
no confirmed exceptions from predicted reactivity known in the moment
to be added when available
to be added when available, antibody released in November 2014.
Plant material: Arabidopsis thaliana Col-0 (wild type) fls2c mutant (described in Nekrasov et al. 2009). Method: Two-week-old seedlings (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, 5% glycerol, 0.5% Triton X-100, 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. Thehe 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-FLS2, 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 was Agrisera goat-anti rabbit IgG HRP-conjugated (AS09 602) at a dilution of 1:5000 diluted in TBS-T containing 5% milk powder 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 3:1 mix of Super Signal West Pico and Femto Chemiluminescent Substrates (Thermo Scientific.
Courtesy of Dr. Elena Petutsching, Georg-August-University Goettingen, Germany
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