Rat Glucokinase (GCK) ELISA Kit

Principle of the Assay

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

Catalyzes the phosphorylation of hexose, such as D-glucose, D-fructose and D-mannose, to hexose 6-phosphate (D-glucose 6-phosphate, D-fructose 6-phosphate and D-mannose 6-phosphate, respectively) (PubMed:6477520, PubMed:12513690, PubMed:24187134). Compared to other hexokinases, has a weak affinity for D-glucose, and is effective only when glucose is abundant (PubMed:6477520). Mainly expressed in pancreatic beta cells and the liver and constitutes a rate-limiting step in glucose metabolism in these tissues (By similarity). Since insulin secretion parallels glucose metabolism and the low glucose affinity of GCK ensures that it can change its enzymatic activity within the physiological range of glucose concentrations, GCK acts as a glucose sensor in the pancreatic beta cell (By similarity). In pancreas, plays an important role in modulating insulin secretion (By similarity). In liver, helps to facilitate the uptake and conversion of glucose by acting as an insulin-sensitive determinant of hepatic glucose usage (By similarity). Required to provide D-glucose 6-phosphate for the synthesis of glycogen (By similarity). Mediates the initial step of glycolysis by catalyzing phosphorylation of D-glucose to D-glucose 6-phosphate (PubMed:6477520, PubMed:12513690).

GENE ID	24385
SWISS PROT	P17712
SYNONYMS	GK; GLK; HHF3; HK4; HKIV; HXKP; MODY2; Hexokinase-D; Hexokinase 4; Maturity Onset Diabetes Of The Young 2

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 GCK were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level GCK 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 0.24ng/mL.
ASSAY RANGE	0.781-50ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of GCK. No significant cross-reactivity or interference between GCK and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between GCK and all analogs, therefore, cross reactivity may still exist.