PIP1;3 | Aquaporin, plasma membrane intrinistic protein 1-3
AS09 505 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Oryza sativa
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KLH-conjugated peptide derived from Oryza sativa PIP1s
26-29 kDa (Oryza sativa)
1.5 µg of crude membrane fraction/lane from Oryza sativa L. cv. Akitakomachi were separated on 12 % SDS-PAGE and blotted 1h to PVDF membrane (40 min. at 10 V using BioRad semidry transfer). Filters were blocked 1h with 5 % low-fat milk powder in TBS-T (0.05% Triton X.100). Membranes were washed 5 times with TBS-T, each time in a fresh polystyrene box and probed with anti-PIP1s antibodies (AS09 505, 1:2000, 1h) and secondary anti-rabbit (1:2000, 1 h). All steps were performed in RT with agitation.
Peptide used to elicti this antibody has been added to primary antibody incubation (right panel, + +) and therefore specific signal has been depleted in this neutralization/immunocompetition assay.
0.1 % sodium azide is added as preservative. For antibody re-suspending information check the tube lable.
Antibodies will detect target protein in a few µg of a crude preparation loaded per well. If purified preparations of vacuolar and plasma membranes are used, one µg load per well should be sufficient.
Protein or membrane sample should be treated at 70°C for 10 min before loading on the gel.
Diluted antibody solution can be used 2 to 3 times within one month if it contains 0.1 % sodium azide as preservative and is stored at -20ºC to -80ºC.
Triton X-100 should not be included in the protein extraction buffer, when cell organelles or membrane proteins must be separated from soluble proteins. Because, Triton X breaks membrane structure and solubilizes most membranes proteins. Furthermore, it should be noted that Triton X at high concentrations binds SDS and mask the detergent effect of SDS for SDS-PAGE. Also, micelles of Triton X behave as a large complex with molecular mass of 90 kDa at high concentrations in SDS-PAGE.
PIP1s - is a plasma membrane aquaporine which facilitates trasport of water across cell membrane.
Liu et al. (2013). Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils. PLoS Genet 9(8): e1003704. doi:10.1371/journal.pgen.1003704 (Oryza sativa, western blot)
Sakurai et al. (2008). Tissue and cell-specific localization of rice aquaporins and their water transport activities- Plant Cell Physiol. 49: 30-39.
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