SRK2E | Ser/Thr-protein kinase SnRK2.6
AS13 2635 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Bacterial lysates were separated on 12% SDS-PAGE and blotted 1h to PVDF using semi-dry or tank transfer. Blots were blocked with 5 % milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 3 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 from Agrisera) diluted to 1:50 000 in for 30 min. at RT with agitation. The blot was washed as above and developed for 3 min with ECL according to the manufacturer's instructions. Exposure time was 30 seconds.
Courtesy of Dr. Agnieszka Ludwików, UAM, Poznań, Poland
Protein A agarose beads (40µl) where coated with 10µl (1µg/ul) antibodies and after incubation with amount of extract (10 mg/ml) indicated washed extensively and loaded on gel. In gel kinase assay was performed as described in Fujii, 2007.
Autoradiograph shows immunoprecipitated kinase from plant extracts. 1 beads with BSA 20 µl loaded on gel 2 beads with plant extract (WT) 20 µl loaded on gel 3 beeds with plant extract (mutant X) 20 µl loaded on gel 4 beads with BSA 10 µl loaded on gel 5 beads with plant extract (WT) 10 µl loaded on gel 6 beeds with plant extract (mutant X) 10 µl loaded on gel 7 beads with BSA 5 µl loaded on gel 8 beads with plant extract (WT) 5 µl loaded on gel 9 beeds with plant extract (mutant X) 5 µl loaded on gel
Courtesy of Dr. Szymon Świeżewski, Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland
Synonyms: OST1, Open stomata 1
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