APX | L-ascorbate peroxidase
AS08 368 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, A.maritima, C. annuum, Citrus sp., D. sanguinalis, E. crus-galli, l. formosana, M. esculenta, M. sativa, N.tabacum, O. sativa, P. miliaceum, S. oleracea, S. superba, S. vulgaris, Triticum aestivum, Z. mays
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Synthetic peptide derived from Arabidopsis thaliana tAPX (acession At1g77490) and sAPX (At4g08390) protein sequences, coupled to BSA. Five out of twelve amino acids are also identical with cAPX1 (At1g07890), cPX2 (At3g09640) and pAPX (At4g35000)
25-38 kDa for A. thaliana
Brassica rapa subsp. oleifera Stromal APX; Glycine max, Glycine soja L-ascorbate peroxidase T, chloroplastic; Medicago truncatula thylakoid-bound APX; Pisum sativum Chloroplast stromal ascorbate peroxidase 12; Solanum lycopersicum thylakoid-bound APX; Spinacia oleracea stromal APX; Theobroma cacao L-APX T isoform 3; Vitis vinifera
5 to 20 μg of total leaf protein from Arabidopsis thaliana (left panel) and chloroplast fractions (thylakoids and soluble, right panel) was separated on 15% polyacrylamide gel with 6M urea and blotted on PVDF. Filters were blocked 1h with 5% BSA, incubated with anti-APX antibody (1: 2000, 1h) followed by incubation with secondary HRP-coupled anti rabbit antibody (1: 10 000, 1h). Signal was detected with chemiluminescence detection reagent. AS08 368 is reactive to thylakoid (tAPX, 38 kDa), stromal (sAPX, 33 kDa), peroxisomal (pAPX, 31 kDa) and cytoplasmic (cAPX1 + cAPX2, 25 kDa) forms of ascorbate peroxidases.
Total proteins of Arabidopsis thaliana leaves were extracted with 10 % TCA and precipitated. The pellet was washed with acetone and resuspended in 100mM Tris-HCl (pH 7.5), 1mM EDTA, 2% (w= v) SDS, 1:100 of protease inhibitor cocktail (Thermo Scientific), 1 mM PMSF. Leaves were also grinded in 100 mM Tris-HCl (pH 7.5), MgCl2 10 mM, 1 mM EDTA, 1 mM PMSF, 1/100 of protease inhibitor cocktail and centrifugated. The supernatant (soluble fraction) was separated and the pellet (membrane fraction) was resuspended in the same buffer with 6 M urea and 1% SDS. Different amounts of proteins were separated in 15 % polyacrylamide gel with 6M urea after denaturation (70°C 5 min) and blotted on PVDF. Filters were blocked 1h with 5% BSA, Incubated with anti-APX antibodies at a dilution 1:2000, 1h/RT, washed 4 times with TBS tween (5 min each) and incubated with HRP coupled anti-rabbit IgG secondary antibody in dilution 1:10 000 1h/RT (AS09 602, Agrisera). After incubation with secondary antibody, the filter was washed 4 times with TBS (5 min each) and signal was detected with chemiluminescent detection reagent (30 secs exposition in film).
Courtesy Manuel Guinea Diaz, University of Turku, Finland
This product can be sold containing proclin if requested
APX plays a key role in plant antioxidant system by reducing hydrogen peroxide to water. Cellular localization includes chloroplast (tAPX and sAPX), cytosol (cAPX) and peroxisome (pAPX).
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