Mouse GCK (Glucokinase) CLIA Kit (MOES00293)

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MOES00293
€649

Description

ELISA Kit Technical ManualMSDS

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.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection methodChemiluminescence
Detection range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Mouse GCK in samples. No significant cross-reactivity or interference between Mouse GCK and analogues was observed.

Typical Data

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.

Concentration
(pg/mL)
RLU Average Corrected
200053105
58337
5572155693
100023121
24827
2397423946
50011234
10830
1103211004
2505148
5440
52945266
1252759
2457
26082580
62.51401
1221
13111283
31.25642
706
674646
027
29
28--

Precision

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
Sample123123
n202020202020
Mean (pg/mL)92.83290.28927.2499.66266.41835.45
Standard deviation9.3928.8091.7012.2629.4973.52
C V (%)10.129.929.8912.3011.078.80

Recovery

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 (%)
Serum (n=5)84-9791
EDTA plasma (n=5)95-110103
Cell culture media (n=5)87-10495

Linearity

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)
1:2Range (%)103-11798-11592-109
Average (%)109105100
1:4Range (%)94-10994-10698-112
Average (%)10199104
1:8Range (%)89-10286-10192-107
Average (%)969299
1:16Range (%)97-108101-11385-98
Average (%)10210790

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.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 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 Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 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 2.7.1.2; 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

UniProt Code:P52792
NCBI GenInfo Identifier:31982798
NCBI Gene ID:103988
NCBI Accession:NP_034422.2
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
Protein Family:Glucokinase
UniProt Gene Name:Gck  
UniProt Entry Name:HXK4_MOUSE
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Additional Information

Reactivity:
Mouse
ELISA Type:
CLIA
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