TIP2;2 | tonoplast intrinistic protein 2-2, C-terminal
AS09 511 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Oryza sativa
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25 | 20-24 kDa
1.5 µg of crude membrane fraction/lane from Oryza sativa L. cv. Akitakomachi leaf blade and root 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-TIP2;2 C-terminal antibodies (AS09 511, 1:1000, 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.
TIP2-2 belongs to aquaporin protein family and facilitates transport of water and small neutral solutes across tonoplast membrane.