Mouse GCK (Glucokinase) CLIA Kit (MOES00293)
Mouse GCK (Glucokinase) CLIA Kit
This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Mouse GCK . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse GCK and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse GCK, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Mouse GCK. The concentration of Mouse GCK in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|Detection range||31.25-2000 pg/mL|
|Sample type||Serum, plasma and other biological fluids|
|Repeatability||CV < 15%|
This kit recognizes Mouse GCK in samples. No significant cross-reactivity or interference between Mouse GCK and analogues was observed.
As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse GCK were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse GCK were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||10.12||9.92||9.89||12.30||11.07||8.80|
The recovery of Mouse GCK spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||95-110||103|
|Cell culture media (n=5)||87-104||95|
Samples were spiked with high concentrations of Mouse GCK and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum (n=5)||EDTA plasma (n=5)||Cell culture media (n=5)|
Kit Components & Storage
An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
|Micro CLIA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100X)||1 vial, 120 uL|
|Concentrated HRP Conjugate (100X)||1 vial, 120 uL||-20'C(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4'C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25X)||1 vial, 30 mL|
|Substrate Reagent A||1 vial, 5 mL||4'C (shading light)|
|Substrate Reagent B||1 vial, 5 mL||4'C (shading light)|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Mouse GCK (Glucokinase) CLIA Kit (MOES00293) Assay procedure
- 1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- 2. Aliquot 100µl of standard solutions into the standard wells.
- 3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
- 5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
- 7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- 8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- 9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
- 11. Determine the RLU value of each well immediately.
Mouse GCK (Glucokinase) CLIA Kit (MOES00293) Protein Information
|UniProt Protein Function:||GK: a glycolytic enzyme that catalyzes glucose metabolism in the liver and pancreatic beta cells. Acts as a ""glucose sensor"" in beta cells. The first and rate-limiting step in glycosis, a pathway that produces energy in the form of ATP from glucose. Glucokinase traps glucose inside the cell by catalyzing its phosphorylation to produce glucose-6-phosphate. Has a lower affinity for glucose than the three other isozymes of hexokinase, allowing other organs such as the brain and muscles to have first call on glucose when its supply is limited. Unlike other hexokinases, glucokinase is not inhibited by glucose-6-phosphate. Glucokinase is found in the outer membrane compartment of mitochondria. May bind VDAC, suppressing mitochondrial function. Glucokinase transcription is induced by insulin, perhaps via the activation of Stat 5B. Mutant glucokinase causes a rare form of diabetes and may also play a role in type 2 diabetes. Three splice variant isoforms of human glucokinase have been described.|
|UniProt Protein Details:|
Protein type:Carbohydrate Metabolism - amino sugar and nucleotide sugar; Kinase, other; Carbohydrate Metabolism - galactose; Mitochondrial; Carbohydrate Metabolism - glycolysis and gluconeogenesis; EC 22.214.171.124; Carbohydrate Metabolism - starch and sucrose
Cellular Component: nucleoplasm; mitochondrion; cell; cytoplasm; actin filament; cell cortex; cytosol; basal cortex; nucleus; secretory granule
Molecular Function:transferase activity; hexokinase activity; protein binding; magnesium ion binding; nucleotide binding; kinase activity; ADP binding; phosphotransferase activity, alcohol group as acceptor; glucokinase activity; protein phosphatase binding; ATP binding; glucose binding
Biological Process: positive regulation of glycogen biosynthetic process; glucose 6-phosphate metabolic process; lipid homeostasis; glucose homeostasis; NADP metabolic process; cellular response to glucose starvation; elevation of cytosolic calcium ion concentration; detection of glucose; negative regulation of gluconeogenesis; second-messenger-mediated signaling; glycogen biosynthetic process; glycolysis; regulation of potassium ion transport; glucose metabolic process; positive regulation of insulin secretion; fructose 2,6-bisphosphate metabolic process; cellular response to insulin stimulus; carbohydrate metabolic process; carbohydrate phosphorylation; positive regulation of glycolysis; negative regulation of epinephrine secretion; cell glucose homeostasis; regulation of insulin secretion; phosphorylation; positive regulation of phosphorylation
|NCBI GenInfo Identifier:||31982798|
|NCBI Gene ID:||103988|
|UniProt Secondary Accession:||P52792,P52791,|
|UniProt Related Accession:||P52792|
|Molecular Weight:||52,089 Da|
|NCBI Full Name:||glucokinase|
|NCBI Synonym Full Names:||glucokinase|
|NCBI Official Symbol:||Gck|
|NCBI Official Synonym Symbols:||Gk; GLK; HK4; HKIV; HXKP; MODY2; Gls006|
|NCBI Protein Information:||glucokinase; HK IV; hexokinase 4; hexokinase-4; hexokinase-D; hexokinase type IV|
|UniProt Protein Name:||Glucokinase|
|UniProt Synonym Protein Names:||Hexokinase type IV; HK IV; Hexokinase-4; HK4; Hexokinase-D|
|UniProt Gene Name:||Gck|
|UniProt Entry Name:||HXK4_MOUSE|