STN8 | Serine/threonine-protein kinase STN8, chloroplastic
AS10 1601 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana, Triticum aestivum
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|Recommended dilution||1: 2000 with standard ECL (WB)|
|Expected | apparent MW||
54.9 kDa or 46 kDa on 6 M urea gel
|Confirmed reactivity||Arabidopsis thaliana, Triticum aestivum
|Predicted reactivity||Oryza sativa|
|Not reactive in||no confirmed exceptions from predicted reactivity known in the moment|
For best results with this antibody sample buffer needs to contain 6 M urea (138mM TrisHCl pH 6.8, 6M urea, 22.2% Glycerol, 4.3% SDS).
STN8 is a nuclear encoded protein which is localized in chloroplast, hence posses a chloroplastic target peptides (cTP) at the beginning of the amino acid sequence which is cut off. Accroding to TargetP (program that predicts the length of the cTP:s) the length of cTP for Stn8 is 49 amino acids. STN8 portein mobility can be also affected by urea present in the gel.
|Selected references||Li et al. (2015). Effect of hydrogen sulfide on D1 protein in wheat under drought stress. Acta Physiologiae Plantarum November 2015, 37:225.
Flood et al. (2014). Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana: is it caused by genetic variation in the STN kinases? Philos Trans R Soc Lond B Biol Sci. 2014 Mar 3;369(1640):20130499. doi: 10.1098/rstb.2013.0499. Print 2014.
Yin et al. (2012). Photosystem II Function and Dynamics in Three Widely Used Arabidopsis thaliana Accessions. PLOS ONE, open access.
Thylakoids (2 or 3 µg of chlorophyll/lane) from Arabidopsis thaliana wide type (WT) and a stn8 mutant were isolated according to Sirpiö et al (2011, Methods Mol Biol. 2011; 775:19-30). Denaturated samples were separated on 12 % SDS-PAGE with 6 M urea and blotted for 1h to a PVDF membrane using a semi-dry transfer. Blots were blocked with 5% milk in TBS for 1h at room temperature (RT) with slow agitation. Blots were incubated in primary antibodies at a dilution of 1: 1 000 overnight at 4°C with slow agitation in 1 % milk/TTBS. Blots were rinsed briefly once, then washed twice for 10 min with TTBS at RT with vigorous agitation. Blots were incubated in secondary antibodies (anti-rabbit IgG horse radish peroxidase conjugated) in 1% milk/TTBS for 2 hours at RT with slow agitation, washed as above and incubated for 5 min with ECL solution according to the manufacturers’ instructions. Exposure time was 2 min. Since the STN8 antibody cross-reacts with LHCII, the lower part of the gel should be cut out before blotting.
Courtesy Virpi Paakkarinen, University of Turku, Finland
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