Western blot analysis of extracts from 293, C6 and mIMCD-3 cells using #3487 AQP2 Antibody.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
AQP2 Antibody detects endogenous levels of total AQP2 protein.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln263 of human AQP2. Antibodies are purified by peptide affinity chromatography.
Aquaporin 2 (AQP2) is a water transport protein that forms water channels in kidney tubules and plays a predominant role in controlling organism water homeostasis (1). Members of the aquaporin family are multiple pass transmembrane proteins that form homotetramers to facilitate the flow of water across the plasma membrane. At least thirteen aquaporins have been indentified to date (AQP0 through AQP12) and together this family of small, hydrophobic proteins plays a role in an array of biological processes that include urine formation, cell motility, fertilization, cell junction formation and regulation of overall water homeostasis (2). AQP2 tetramers form water channels that facilitate water transport and excretion in the kidney (3). This transport protein is localized to the plasma membrane is response to endocrine signaling. Posterior pituitary hormones arginine vasopressin (AVP) and ADH regulate osmotic water cell permeability by triggering phosphorylation and subsequent exocytosis of AQP2 (1,4). Mutations in the corresponding AQP2 gene cause a rare form of diabetes known as nephrogenic diabetes insipidus. This autosomal dominant disorder is characterized by abnormal water reabsorption by kidney tubules due, in part, to either nonfunctional or mislocalized AQP2 protein (5).
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