Elip | Early light induced protein
AS06 147A | Clonality: Polyclonal | Host: Rabbit | Reactivity: A.thaliana, Ch. reinhardtii, P. sativum, Vitis sp.
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20 kDa (Pisum sativum)
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Arabidopsis thaliana leaf extract (50 mg leaf material was ground with the grinding buffer (2% lithium dodecyl sulfate, 0.35 M sucrose, 100 mM dithiothreitol, 50 mM Tris HCl pH 8,0) ) 10 µl: Arabidopsis plants were exposed to strong light 1000 µmol photons m-2 s-1 for 24 hours at 6 degrees. 2. Arabidopsis leaf extract 10 µl: Arabidopsis plants were exposed to strong light 1000 umol photons m-2 s-1 for 24 hours at 25°C. Extraction methods: 50 mg leaf material was ground with the grinding buffer (2% lithium dodecyl sulfate, 0.35 M sucrose, 100 mM dithiothreitol, 50 mM Tris HCl pH 8). No denaturing step: Extracts were centrifuged for 5 min at 15 000 rpm using a microcentrifuge. 10 µl of protein extracts per lane was resolved on 14% SDS-PAGE. Proteins were blotted on PVDF membranes using wet (tank) transfer. Blots were blocked with blocking buffer (PBS containing 0.2% tween and 4% skim milk) for 1 hour. Blots were incubated in the primary antibody solution (1:5000) for 1 hour at 25°C. Blots were rinsed with PBS-T for 5 min, and the rinsing steps were repeated four times. The secondary antibody (anti-rabbit IgG-conjugated HRP) diluted 20 000 times. Blots were rinsed with PBS-T for 5 min, and the rinsing steps were repeated four times. Finally, blots were incubated with Western Lightning Plus (Perkin Elmer) Exposed with a CCD camera-equipped detector for 30 min.
Courtesy Dr. Ryouichi Tanaka, Hokkaido University, Japan
About 30 µg of total protein from Chlamydomonas reinhardtii, extracted with pre-cooled buffer (10% glycerol, 20 mM HEPES, 5 mM MgCl2, 2.5 mM EDTA, 10 mM KCl, 1 mM PMSF, 0,5% DTT; pH=7,4) and denatured with SDS/mercaptoethanol loading buffer at 95C for 5 min, were separated on 12% SDS-PAGE and blotted at 60V for 2,5h to nitrocellulose membrane using tank transfer. Blots were blocked with 0.2% Tween-20 in TBS (overnight, at 5OC) with agitation. Blot was incubated in the primary antibody at a dilution of 1:500 in TBS-T (0.05% Tween-20 in TBS) overnight at 5OC with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from goat) diluted 1:15 000 in TBS-T, for 2h at RT with agitation. The blot was washed as above and developed using *DAB/H2O2 mixture in TBS for approximately 5 min.
* DAB = 3,3'-Diaminobenzidine
Courtesy of Dr. Anna Aksmann, Department of Plant Physiology and Biotechnology, University of Gdańsk, Poland
Min. 30 µg of total protein has to be loaded per lane. Please note that level of Elip proteins varies considerably between different plant species and therefore protein load/well has to be adjusted.
Early light-induced proteins (ELIPs) are light stress-induced proteins related to the chlorophyll a/b binding protein family from higher plants and green algae located in the thylakoid membranes and involved in photosynthesis.
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