Mouse GCK (Glucokinase) ELISA Kit (MOES01076)

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MOES01076
€549

Description

ELISA Kit Technical ManualMSDS

Mouse GCK (Glucokinase) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA 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 ELISA 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 blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse GCK. The concentration of Mouse GCK in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection MethodColormetric
Detection Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

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 OD 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
(ng/mL)
O.D Average Corrected
102.195
2.207
2.2012.128
51.513
1.515
1.5141.441
2.50.879
0.855
0.8670.794
1.250.451
0.471
0.4610.388
0.630.224
0.194
0.2090.136
0.320.168
0.156
0.1620.089
0.160.119
0.121
0.120.047
00.073
0.073
0.073--

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 (ng/mL)0.480.954.990.520.935.39
Standard deviation0.030.050.180.030.050.19
C V (%)6.255.263.615.775.383.53

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)85-10092
EDTA plasma (n=5)89-10196
Cell culture media (n=5)90-10498

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 (%)95-10999-11486-97
Average (%)10210691
1:4Range (%)92-10584-9888-101
Average (%)979195
1:8Range (%)91-10186-9986-98
Average (%)969192
1:16Range (%)85-10185-10182-94
Average (%)929287

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 ELISA 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 Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Mouse GCK (Glucokinase) ELISA Kit (MOES01076) 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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Mouse GCK (Glucokinase) ELISA Kit (MOES01076) 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:
Sandwich
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