For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of mouse AQP4x protein. Antibodies are purified by protein A and peptide affinity chromatography.
Aquaporins (AQP) are integral membrane proteins that serve as channels in the transfer of water and small solutes across the membrane. There are 13 isoforms of AQP that express in different types of cells and tissues (1,2). AQP1 is found in blood vessels, kidney, eye, and ear. AQP2 is found in the kidney, and it has been shown that the lack of AQP2 results in diabetes (1,3). AQP4 is present in the brain, where it is enriched in astrocytes (1,2,4). AQP5 is found in the salivary and lacrimal gland, AQP6 in intracellular vesicles in the kidney, AQP7 in adipocytes, AQP8 in kidney, testis, and liver, AQP9 is present in liver and leukocytes, and AQP10-11 in the intestine (1,3,4). AQPs are essential for the function of cells and organs. It has been shown that AQP1 and AQP4 regulate the water homeostasis in astrocytes, preventing cerebral edema caused by solute imbalance (5). Several studies have shown the involvement of AQPs in the development of inflammatory processes, including cells of innate and adaptive immunity (6,7).
The mouse AQP4x has an extended sequence at the C-terminal (ex) as a result of a codon readthrough during alternative splicing (8). The C-terminal extension in the mouse AQP4x changes the localization to a more perivascular polarization (8). Programmed translational readthrough could occur also in human AQP4x (9).
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