HY5 | Protein long hypocotyl 5

AS12 1867 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana

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

Not reactive in

Hordeum vulgare, Oryza sativa, Pisum sativum, Populus sp. , Solanum lycopersicum, Triticum aestivum

Application examples

Application examples Application information

Western blot using anti-HY5 antibodies

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

western blot using anti-HY5 antibodies

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).

Related products

Related products Collection of antibodies to proteins involved in photomorphogenesis

Plant protein extraction buffer

Secondary antibodies

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.