Mouse HK1 (Hexokinase 1) ELISA Kit (MOES01175)
- Product type:
- ELISA Kit
- ELISA Type:
|Detection Range:||31.25-2000 pg/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Mouse HK1 in samples. No significant cross-reactivity or interference between Mouse HK1 and analogues was observed.|
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 HK1. 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 HK1 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 HK1, 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 HK1. The concentration of Mouse HK1 in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||HK1: a glycolytic enzyme that catalyzes the reaction ATP + D-hexose = ADP + D-hexose 6-phosphate. The first and rate-limiting step in glycosis, a pathway that produces energy in the form of ATP from glucose. An allosteric enzyme inhibited by its product glucose-6-phosphate (Glc-6-P). HK-2 and its mitochondrial receptor (VDAC) play the most pivotal and direct roles in the ""Warburg effect"". Acts as a ""glucose sensor"" by trapping glucose inside the cell by catalyzing its phosphorylation to produce Glc-6-P. In vertebrates there are four major glucose-phosphorylating isoenzymes, designated hexokinase I, II, III and IV (glucokinase). Four human isoforms are produced by alternative splicing and alternative initiation. Isoform HK1 is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. HK1 is present in most tissues but is especially prominent in brain and kidney. Isoform HK1-SC is is an integral membrane protein detected in round spermatids, condensing spermatids and mature sperm where it is found in the head membranes, mitochondria of the midpiece and the fibrous sheath of the flagellum. Isoform HK1-SA is first expressed during meiosis and continues to be present in postmeiotic germ cells while isoform HK1-SB is present only in postmeiotic germ cells.|
|UniProt Protein Details:|
Protein type:Carbohydrate Metabolism - amino sugar and nucleotide sugar; Carbohydrate Metabolism - starch and sucrose; EC 2. 7. 1. 1; Mitochondrial; Carbohydrate Metabolism - fructose and mannose; Carbohydrate Metabolism - galactose; Kinase, other; Carbohydrate Metabolism - glycolysis and gluconeogenesis
Cellular Component: mitochondrial outer membrane; protein complex; membrane; mitochondrion; cell; caveola; cytosol; cilium; lipid raft
Molecular Function:hexokinase activity; protein homodimerization activity; nucleotide binding; glucokinase activity; protein kinase activity; transferase activity; protein binding; mannokinase activity; protein complex binding; fructokinase activity; kinase activity; phosphotransferase activity, alcohol group as acceptor; catalytic activity; ATP binding; glucose binding
Biological Process: peptidyl-tyrosine phosphorylation; glycolysis; metabolic process; protein amino acid autophosphorylation; glucose 6-phosphate metabolic process; regulation of anion channel activity; peptidyl-threonine phosphorylation; protein amino acid phosphorylation; peptidyl-serine phosphorylation; carbohydrate phosphorylation; carbohydrate metabolic process; cell glucose homeostasis; phosphorylation; negative regulation of apoptosis
|NCBI GenInfo Identifier:||341941063|
|NCBI Gene ID:||15275|
|NCBI Accession:||P17710. 3|
|UniProt Secondary Accession:||P17710,Q61659, Q64476, Q64479, E9PXQ3,|
|UniProt Related Accession:||P17710|
|Molecular Weight:||17. 7kDa|
|NCBI Full Name:||Hexokinase-1|
|NCBI Synonym Full Names:||hexokinase 1|
|NCBI Official Symbol:||Hk1|
|NCBI Official Synonym Symbols:||dea; Hk-1; Hk1-s; mHk1-s; BB404130|
|NCBI Protein Information:||hexokinase-1; HK I; downeast anemia; hexokinase type I; hexokinase, tumor isozyme|
|UniProt Protein Name:||Hexokinase-1|
|UniProt Synonym Protein Names:||Hexokinase type I; HK I; Hexokinase, tumor isozyme|
|Protein Family:||Probable histidine kinase|
|UniProt Gene Name:||Hk1|
|UniProt Entry Name:||HXK1_MOUSE|
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse HK1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse HK1 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||6.27||4.24||3.53||6.33||5.74||3.27|
The recovery of Mouse HK1 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)||90-105||98|
|Cell culture media (n=5)||95-108||100|
Samples were spiked with high concentrations of Mouse HK1 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)|
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 ELISA Plate(Dismountable)||8 wells ×12 strips||-20°C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 µL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 µL||-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 (25×)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4°C(shading light)|
|Stop Solution||1 vial, 10 mL||4°C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.