Western blot analysis of extracts from A-431 cells, untreated (-) or treated with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml, 2 min; +), and HeLa cells, untreated (-) or pervanadate-treated (1 mM, 15 min; +), using Phospho-Na,K-ATPase α1 (Tyr10) (E1Y9C) Rabbit mAb (upper) and Na,K-ATPase Antibody #3010 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
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
Phospho-Na,K-ATPase α1 (Tyr10) (E1Y9C) Rabbit mAb recognizes endogenous levels of Na,K-ATPase α1 protein only when phosphorylated at Tyr10. The antibody cross-reacts with an induced 75-80 kDa doublet of unknown origin.
Rat, Bovine, Dog, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr10 of human Na,K-ATPase α1 protein.
The Na,K-ATPase is an integral membrane heterodimer belonging to the P-type ATPase family. This ion channel uses the energy derived from ATP hydrolysis to maintain membrane potential by driving sodium export and potassium import across the plasma membrane against their electrochemical gradients. It is composed of a catalytic α subunit and a β subunit (reviewed in 1). Several phosphorylation sites have been identified for the α1 subunit. Tyr10 is phosphorylated by an as yet undetermined kinase (2), Ser16 and Ser23 are phosphorylated by PKC, and Ser943 is phosphorylated by PKA (3-5). All of these sites have been implicated in the regulation of enzyme activity in response to hormones and neurotransmitters, altering trafficking and kinetic properties of Na,K-ATPase. Altered phosphorylation in response to angiotensin II stimulates activity in the rat proximal tubule (6). Na,K-ATPase is also involved in other signal transduction pathways. Insulin regulates its localization in differentiated primary human skeletal muscle cells, and this regulation is dependent on ERK1/2 phosphorylation of the α subunit (7). Na,K-ATPase and Src form a signaling receptor complex that affects regulation of Src kinase activity and, subsequently, its downstream effectors (8,9).
Explore pathways + proteins related to this product.
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