HY5 | Protein long hypocotyl 5
AS12 1867 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Hordeum vulgare, Oryza sativa, Pisum sativum, Populus sp. , Solanum lycopersicum, Triticum aestivum
10 µg of total protein extracted freshly from 7-d old Arabidopsis thaliana seedlings using Trichloroacetic acid and Acetone (Mechin et al. 2007), and denatured with LDS (Lithium dodecyl sulfate) sample buffer at 70°C for 10 min. Proteins were separated on 12% SDS-PAGE and blotted 7 min to PVDF (pore size of 0.2 um), using semi-dry transfer. Blot was blocked with 5% milk for 4°C/ON with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 2 h/RT with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly, then washed three times for 15 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:10,000 in for 1 h/RT with agitation. The blot was washed as above and developed for 2 min with chemiluminescence detection reagent. Exposure time was 100 seconds.
The seedlings were grown 4 d in dark and 3 d in continuous light (~120 uE). Seedlings were ground in whole for protein extraction.
Courtesy of Xin Hou, Pogson Lab, Research School of Biology, ANU College of Science, Australia
20 ug of total protein from control (1), 35S::YFP-HY5-HA (2, red arrow), 35S::YFP-HY5-HA + 35S::CFP-X protein (green arrow), were separated on 12 % SDS-PAGE using tank transfer and blotted 1 h to PVDF (Biorad). Blots were blocked with 5 % skim milk for 1h at room temperature (RT) with agitation. Blot was incubated in the anti-HY5 antibody (second panel from the left) at a dilution of 1: 1000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min. in PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG, HRP conjugated from Agrisera, AS09 602), diluted to 1: 10 000 for 1h at RT with agitation. The blot was washed as above and developed for 5 min. with chemiluminescent detection, according to the manufacturer's instructions. Exposure time was 60 seconds.
Courtesy of Dr. Seok Keun Cho, University of Copenhagen, Danmark
Lee et al. (2017). The F-box protein FKF1 inhibits dimerization of COP1 in the control of photoperiodic flowering. Nat Commun. 2017 Dec 22;8(1):2259. doi: 10.1038/s41467-017-02476-2.
Sinclair et al. (2017) Etiolated Seedling Development Requires Repression of Photomorphogenesis by a Small Cell-Wall-Derived Dark Signal. Curr Biol. 2017 Nov 20;27(22):3403-3418.e7. doi: 10.1016/j.cub.2017.09.063.
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