STN7 | Serine/threonine-protein kinase STN7, chloroplastic
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|Recommended dilution||1: 2000 with standard ECL (WB)|
|Expected | apparent MW||
63.2 | 44 kDa (on urea gel), 55 kDa (no urea)
|Confirmed reactivity||Arabidopsis thaliana, Nicotiana tabacum|
|Predicted reactivity||Glycine max, Oryza sativa, Ricinus communis, Zea mays, Vitis vinifera
|Not reactive in||Chlamydomonas reinhardtii|
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).
STN7 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 Stn7 is 45aa. STN7 portein mobility can be also affected by urea present in the gel.
|Selected references||Mekala et al. (2015). Plants actively avoid state-transitions upon changes in light intensity - role of light-harvesting complex II protein dephosphorylation in high light. Plant Physiol. 2015 Apr 22. pii: pp.00488.2015.
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 3 ug of chlorophyll/lane from Arabidopsis thaliana were extracted according to Sirpiö et al (2011, Methods Mol Biol. 2011;775:19-30) and were separated on 12 % SDS-PAGE containing 6 M urea and blotted 1h to PVDF. Blots were blocked with 5% milk in TTBS for 1h at room temperature (RT) with slow agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 over night at 4°C with slow agitation in 1 % milk TTBS . The antibody solution was decanted and the blot was rinsed briefly only once, then washed once for 15 min only in TBS-T at RT with vigorous agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated ) diluted to 1:10 000 in 1% milk in TTBS for 2h at 4°C with slow agitation. The blot was washed as above and incubated for 5 min with ECL-solution according to the manufacturers instructions. Exposure time was 1 min.
To avoid cross-reactivity with LHC proteins a gel can be run further to let proteins below 25 kDa leave a gel.
Courtesy Dr. Maija Holmström, University of Turku, Finland
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