HY5 | Protein long hypocotyl 5
AS12 1867 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Product Information
Immunogen
KLH-conjugated peptide, derived from Arabidopsis thaliana HY5 protein sequence, UniProt:O24646 , TAIR: AT5G11260. Chosen peptide is not conserved in HY5 protein sequence.
Host
Rabbit
Clonality
Polyclonal
Purity
Affinity purified serum in PBS, pH 7.4
Format
Lyophilized
Quantity
50 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 to 1 : 1000 (WB)
Expected | apparent MW
18.5 kDa
Reactivity
Confirmed reactivity
Arabidopsis thaliana
Predicted reactivity
Brassica pekinensis
Species of your interest not listed? Contact us
Species of your interest not listed? Contact us
Not reactive in
Hordeum vulgare, Oryza sativa, Pisum sativum, Populus sp. , Solanum lycopersicum, Triticum aestivum
Application examples
Application examples
Application information
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
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
Additional information
This antibody detects recombinant HY5 in Nicotiana benthamiana.
Extraction and loading buffer with 6-8 M urea buffer needs to be used when working with endogenous extract to allow detection with this antibody or TCA/acetone protein extraction or as described in Mechin et al. (2007).
Background
Background
HY5 (Protein long hypocotyl 5) is a light-regulated transcription factor that promotes photomorphogenesis in light. Involved in the blue light specific pathway. Heterodimer. Expressed in root, hypocotyl, cotyledon, leaf, stem and floral organs. Localized in nucleus. Alternative names: Protein LONG HYPOCOTYL 5, bZIP transcription factor 56, AtbZIP56.
Product citations
Selected references
Cazzonelli et al. (2019). A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development. https://doi.org/10.1101/528331
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.
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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