STN8 | Serine/threonine-protein kinase STN8 (chloroplastic)

Application example




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

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.

Due to a high background signal with LHCII it is adviced to cut off a membrane below 30 kDa marker.