TIP1;1, TIP1;2 | tonoplast intrinistic protein 1-1, 1-2 (gamma)

7.5 µg / 37.5 µg and 75 µg of total protein extracted freshly isolated from whole leaf extracts o Arabidopsis thaliana wt plants. Material was grinded frozen and then mixed 1:4 with cold isolation buffer (20 mM Tris pH 7.6, 80 mM NaCl, 0.7 mM EDTA, 5 mM MgCl, 1 mM DTT and 2 % SDS). Protein concentration was measured by Bradford and sample was mixed 1:1 with loading dye (Bromphenolblue with 4 %SDS and 2 % bet-mercaptoethanol). Sample was denaturized at 96 °C for 10 min. Samples were separated on12.5% SDS-PAGE and blotted for 45 min to nitrocellulose, using: semi-dry transfer. Blot was blocked with 5 % milk or for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1.000 in TBS-T for ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, As09 602, Agrisera) diluted to 1: 25.000 in for 1 h at RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AgriseraBright (AS16 ECL-N-10). 

Courtesy of Dr. Andras Bittner, IZMB - Zellbiologie der Pflanzen, University of Bonn, Germany

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