TIP1;1, TIP1;2 | tonoplast intrinistic protein 1-1, 1-2 (gamma)
AS09 493 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Raphanus sativus
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KLH-conjugated synthetic peptide conserved in Raphanus sativus TIP1;1 and TIP1;2 (protein accesion number available in Suga et al. 2001). Peptide is also conserved in Arabidopsis thaliana TIP1-1 P25818, At2g36830, TIP1-2 Q41963, At3g26520
25.8 | 23 kDa
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-TIP1s antibodies (AS09 473, 1:1000, 1h) and secondary anti-rabbit (1:2000, 1 h). All steps were performed in RT with agitation.
TIP proteins usually show a faint band of dimeric form at 42 kDa in addition to the major monomer band of 23 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.
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.
TIPs belong to MIP/aquaporin protein family. Alternative names: Aquaporin TIP1-1,gamma-tonoplast intrinsic protein, gamma-TIP, aquaporin TIP, gamma-tonoplast intrinsic protein, gamma-TIP, aquaporin TIP, tonoplast intrinsic protein, root-specific RB7, gamma-tonoplast intrinsic protein 2, gamma-TIP2, salt stress-induced tonoplast intrinsic protein
Suga et al. (2001). Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporings in radish organs. Planta 212:294-304.