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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KLH-conjugated synthetic peptide conserved in Arabidopsis thaliana: PIP1;1 UniProt: P61837,At3g61430 PIP1;2 UniProt: Q06611,TAIR: At2g45960 PIP1;3 UniProt: Q08733,TAIR: At1g01620 , PIP1;4 UniProt: Q39196,TAIR: At4g00430, PIP1;5 UniProt: Q8LAA6 TAIR:At4g23400
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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 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 chemiluminescence reagent according to manufacture instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.
The background at the top of the membrane can be optimized.
Courtesy of Dr. Rosario Vera, UNAM, Mexico
Protein or membrane sample should be treated at 70°C for 10 min before loading on the gel.
PIPs proteins are aquaporins which facilitate the transport of water and small neutral molecules across cell membrane. Alternative names: PIP1-1, PIP1-2, PIP1-3, plasma membrane intrinistic protein 1a, 1b, 1c, PIP1a, PIP1b, PIP1c, plasma membrane aquaporin-1, transmembrane protein A, TMP-A, AthH2, transmembrane protein B, TMP-B,
Related products: PIP (PIP1;1, PIP1;2, PIP1;3, PIP1;4, PIP1;5) | Aquaporins
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