PIP2;1+PIP2;2 | Aquaporin PIP2;1+PIP2;2
AS09 488 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, O. sativa, R. sativus
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30.34 | 28 kDa (Raphanus sativus)
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1 µg and 10 µg of crude membrane fraction/lane from Arabidopsis thaliana 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-PIP2;1 antibodies (AS09 488, 1:1000, 1h) and secondary anti-rabbit (1:2000, 1 h). All steps were performed in RT with agitation.
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.
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.
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.
Peptide used to elicit this antibody is also conserved in Arabidopsis thaliana PIP2;1,PIP2;2, PIP2;3 and this antibody reacted also with PIP2;2 in experiments with knock out plants.
PIPs proteins are aquaporins which facilitate the transport of water and small neutral molecules across cell membrane.
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