Anti-PIP2;2 | Plasma membrane aquaporin 2b

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-PIP2;2 antibodies (AS09 490, 1:1000, 1h) and secondary anti-rabbit (1:2000, 1 h). All steps were performed in RT with agitation.

PIP proteins usually show a faint band of dimeric form at 55 kDa addition to the major monomer band of 28 kDa even in the presence of SDS.

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.

Antiboy will weakly react with PIP2;1 and theoretically with PIP2;3, however this protein is very scarcely expressed in plant tissues. The mRNA content is 1 % of that for PIP2;2, therefore reactivity to PIP2;3 was neglegible in our experiments.

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.