ZEP | Zeaxanthin Epoxidase (anti-peptide)
AS08 289 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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73.8 | 70-75 kDa (Arabidopsis thaliana)
diatoms, Glycine max, Hordeum vulgare, Spinacia oleracea
10 µg of total protein from samples such as Arabidopsis thaliana leaf (1) , Hordeum vulgare leaf (2) were extracted with Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and keept on ice before loading. Protein samples were separated on 4-12% Bolt Plus gels, LDS-PAGE and blotted for 70 minutes to PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent (GE RPN 2125; Healthcare) or 5% non-fat milk dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS10 1489, Agrisera) diluted to 1:25 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with TMA-6 (Lumigen) detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 3 minutes.
Zeaxanthin Epoxidase (ZEP) catalyses the O2-/NAPDHdependent epoxidation of zeaxanthin to violaxanthin via antheraxanthin at the stromal side of the thylakoid membrane of green plants. ZEP also functions in the first step of the biosynthesis of the abiotic stress hormone abscisic acid (ABA). Synonymes:Protein ABA Deficient 1, AtABA1, Protein Impaired in BABA-Induced Sterility 3, Protein Low Expression of Osmotic Stress-Responsive Genes 6, Protein Non-Photochemical Quenching 2.
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