PIP (PIP1;1, PIP1;2, PIP1;3, PIP1;4, PIP1;5) | Aquaporins
AS09 487 | clonality: polyclonal | host: rabbit | reactivity: A. thaliana, J. curcas, P. nigra, P. trichocarpa, R. sativus
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|Recommended dilution||1 : 1000 (WB)|
|Expected | apparent MW||
30.68 | 28 kDa
|Confirmed reactivity||Arabidopsis thaliana, Brassica sp., Jatropha curcas L. cv. "Biji Jarak ", Mesembryantheum crystallinum, Populus nigra, Populus trichocarpa, Raphanus sativus, Thellungiella salsuginea
|Predicted reactivity||Brassica sp., Hordeum vulgare, Juglans regia, Oryza sativa, Populus tremula, Triticum aestivum, Vicia faba|
|Not reactive in||Fragaria sp.|
Protein or membrane sample should be treated at 70°C for 10 min before loading on the gel.
|Selected references||Lopez et al. (2013). Aquaporins And Leaf Hydraulics, Poplar Sheds New Light. Plant Cell Physiol. Sep 20.
Jang et al. (2013). Twoaquaporins of Jatropha are regulated differentially during drought stress and subsequent recovery. J Plant Physiol. March 25.
10 µg of Arabidopsis thaliana tonoplast fraction (1), Thellungiella salsuginea tonoplast fraction (2), Mesembryanthemum crystallinum tonoplast fraction (3), Nicotiana tabacum tonoplast fraction (4), Arabidopsis thaliana plasma membrane fraction (5), Thellungiella salsuginea plasma membrane fraction (6), Mesembryanthemum crystallinum plasma membrane fraction (7), Arabidopsis halleri microsome fraction (8), Brassica sp. microsomal fraction (9) 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 AS09 602,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 Luminata detection reagent according the manufacturers instructions (Millipore). Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.
Courtesy of Dr. Rosario Vera, UNAM, Mexico
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