PIP2;1, PIP2;2, PIP2;3 | Plasma membrane intrinistic protein 2-1,2-2,2-3

Application example

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-PIP2s antibodies (AS09 491, 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.

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.

PIP2;2 is a plasma membrane aquaporin.
Alternative names of isoforms: aquaporin PIP2-1, plasma membrane intrinsic protein 2a, PIP2a, aquaporin PIP2-2, plasma membrane intrinsic protein 2b, PIP2b, TMP2b, Aquaporin PIP2-3, plasma membrane intrinsic protein 2c, PIP2c, TMP2C, RD28-PIP, water stress-induced tonoplast intrinsic protein, (WSII-TIP)