PIP2;1 | Aquaporin PIP2;1 (Oryza sativa)
AS19 4328 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Oryza sativa
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30 | 26-29 kDa
0.5 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-PIP2;1 antibodies (AS19 4328, 1:1000, 1h) and secondary anti-rabbit (1:2000, 1 h). All steps were performed in RT with agitation. Detection was performed using chemiluminescence, according to manufacture's instructions.
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. Antibody is compatible with both, TBS and PBS buffers.For the major component of membranes, such as vacuolar and plasma membrane aquaporins (TIPs and PIPs) and H+-pyrophosphatase, a small amount of the crude membrane fraction (a few micrograms) is enough to detect the antigen protein. When you use the purified preparation of vacuolar and plasma membranes, the antibodies can detect the antigen protein in one microgram membrane preparation.
PIPs proteins are aquaporins which facilitate the transport of water and small neutral molecules across cell membrane.
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