Lhcb2 | LHCII type II chlorophyll a/b-binding protein
AS01 003 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Photosynthetic eukaryotes including A. thaliana, A. hypogaea, C. quitensis Kunt Bartl, C. sativa, H. vulgare, Ch. reinhardtii, L. esculentum (Solanum lycopersicon), M. crystallinum, N. tabacum, O. sativa, P. patens, P. sativum, P. vulgaris, S. alba, S. oleracea, T. aestivum, Triticale, Z. mays
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BSA-conjugated synthetic peptide derived from a highly conserved sequence of Lhcb2 proteins from angiosperms (monocots and dicots) and gymnosperms, including Arabidopsis thaliana Lhcb2.1 UniProt: Q9SHR7, TAIR: AT2G05100, Lhcb2.2 UniProt: Q9S7J7, TAIR:AT2G05070, Lhcb2.3 UniProt:Q9XF87, TAIR:AT3G27690
28.6 | 25 kDa for Arabidopsis thaliana
Species and variants: Pea – Pisum sativum L. Bean – Phaseolus coccineus L. 3h – 3 hours of cold exposure 9h – 9 hours of cold exposure 12h – 12 hours of cold exposure 2d – 2 days of cold exposure
Samples of isolated thylakoids containing 3 µg of chlorophyll were denatured with Laemmli buffer (1 vol : 1 vol) at 75 °C for 5 min. Denatured samples containing 1 µg of chlorophyll were loaded in the gel wells, separated on 12% SDS-PAGE gels and blotted for 45 min at 100 V to PVDF membrane using wet transfer. Blot was blocked with 5% milk in TBS-T for 60 min at room temperature (RT) with agitation. The blot was incubated with the primary antibody at a dilution of 1:500 in 1% Amersham™ ECL Prime Blocking Agent in TBS-T overnight at 4ºC with agitation. The antibody solution was decanted and the blot was washed 3 times for 5 min in TBS-T at RT with agitation. The blot was incubated using a secondary antibody (goat anti-rabbit IgG HRP conjugated, from Agrisera, AS09 602) diluted to 1: 25 000 in 1% milk in TBS-T for 1h at RT with agitation. The blot was washed 5 times for 5 min in TBS-T, 1 time for 5 min in TBS, 1 time for 5 min in 0.1 M Tris (pH 8.5), and developed for 4 min in substrates (0.188 mM coumaric acid, 1.25 mM luminol, 0.01% H2O2). Exposure time was 5 seconds in ChemiDoc scanner (BioRad).
Msc Małgorzata Krysiak, Faculty of Biology, University of Warsaw, Poland
Immunoprecipitation has been done using Immunoprecipitation kit from Roche, Cat.No. 11 719 386 001.
Protein is processed into mature form (Jansson 1999).
The major light-harvesting antenna complex II (LHCII) in photosynthetic eukaryotes is located in the thylakoid membrane of the chloroplast. It is a heterotrimeric complex formed by up to 3 different individual subtypes of chlorophyll a/b-binding proteins: Lhcb1, Lhcb2, and Lhcb3. Lhcb2 is often coded by several nuclear genes and is found together with Lhcb1 within the mobile LHCII trimers involved in state1-state2 transition.
A molecular characterisation of the LHCII proteins can be found in Caffarri et al. (2004) A Look within LHCII: Differential Analysis of the Lhcb1−3 Complexes Building the Major Trimeric Antenna Complex of Higher-Plant Photosynthesis. Biochemistry 43 (29): 9467–9476.
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