Rat ATPase, H+/K+ Exchanging Alpha Polypeptide (ATP4a) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to ATP4a. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to ATP4a. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain ATP4a, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of ATP4a in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

The catalytic subunit of the gastric H(+)/K(+) ATPase pump which transports H(+) ions in exchange for K(+) ions across the apical membrane of parietal cells (PubMed:20921224, PubMed:22448261). Uses ATP as an energy source to pump H(+) ions to the gastric lumen while transporting K(+) ion from the lumen into the cell (PubMed:20921224, PubMed:22448261). Remarkably generates a million-fold proton gradient across the gastric parietal cell membrane, acidifying the gastric juice down to pH 1 (By similarity). Within a transport cycle, the transfer of a H(+) ion across the membrane is coupled to ATP hydrolysis and is associated with a transient phosphorylation that shifts the pump conformation from inward-facing (E1) to outward-facing state (E2). The release of the H(+) ion in the stomach lumen is followed by binding of K(+) ion converting the pump conformation back to the E1 state (PubMed:20921224, PubMed:22448261) (By similarity).

GENE ID	24216
SWISS PROT	P09626
SYNONYMS	Gastric H,K-ATPase Alpha Subunit; H(+)-K(+)-ATPase Alpha Subunit; Proton Pump; Potassium-transporting ATPase alpha chain 1

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level ATP4a were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level ATP4a were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 0.055ng/mL.
ASSAY RANGE	0.156-10ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of ATP4a. No significant cross-reactivity or interference between ATP4a and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between ATP4a and all analogs, therefore, cross reactivity may still exist.