Human Protein Kinase B Gamma (PKBg) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to PKBg. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to PKBg. 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 PKBg, 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 PKBg 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

AKT3 is one of 3 closely related serine/threonine-protein kinases (AKT1, AKT2 and AKT3) called the AKT kinase, and which regulate many processes including metabolism, proliferation, cell survival, growth and angiogenesis. This is mediated through serine and/or threonine phosphorylation of a range of downstream substrates. Over 100 substrate candidates have been reported so far, but for most of them, no isoform specificity has been reported. AKT3 is the least studied AKT isoform. It plays an important role in brain development and is crucial for the viability of malignant glioma cells. AKT3 isoform may also be the key molecule in up-regulation and down-regulation of MMP13 via IL13. Required for the coordination of mitochondrial biogenesis with growth factor-induced increases in cellular energy demands. Down-regulation by RNA interference reduces the expression of the phosphorylated form of BAD, resulting in the induction of caspase-dependent apoptosis.

GENE ID	10000
SWISS PROT	Q9Y243
SYNONYMS	PKBG; PRKB-G; AKT3; RAC-PK-Gamma; RAC-Gamma; STK2; RAC-Gamma Serine/Threonine-Protein Kinase; V-Akt Murine Thymoma Viral Oncogene Homolog 3

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 PKBg were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level PKBg 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 0.56ng/mL.
ASSAY RANGE	1.56-100ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of PKBg. No significant cross-reactivity or interference between PKBg and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between PKBg and all analogs, therefore, cross reactivity may still exist.