Elip | Early light induced protein
AS06 147A | Clonality: Polyclonal | Host: Rabbit | Reactivity: A.thaliana, Ch. reinhardtii, P. sativum, Vitis sp.
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Product Information
Immunogen
KLH-comjugated synthetic peptide derived from known plant ELIP sequnces including Pisum sativum P11432
Host
Rabbit
Clonality
Polyclonal
Purity
Affinity purified serum in PBS pH 7.4
Format
Lyophilized
Quantity
100 µg
Reconstitution
For reconstitution add 50 µl of sterile water.
Storage
Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications
Western blot (WB)
Recommended dilution
1 : 500-1 : 1000 (WB)
Expected | apparent MW
20 kDa (Pisum sativum)
Reactivity
Confirmed reactivity
Arabidopsis thaliana, Chlamydomonas reinhardtii, Pisum sativum, Vitis sp,
Predicted reactivity
Hordeum vulgare, Nicotiana tabacum, Oryza sativa
Application examples
Application examples
Application example
Arabidopsis thaliana leaf extract (see below for extraction method) 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 (see below for extraction method) 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
Arabidopsis thaliana leaf extract (see below for extraction method) 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 (see below for extraction method) 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
Additional information
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.
Background
Background
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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