Human Metabolism ELISA Kits
Human PKM2 (Pyruvate Kinase, Muscle) CLIA Kit (HUES00639)
- SKU:
- HUES00639
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 9.38pg/mL
- Range:
- 15.63-1000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Metabolism
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 15.63-1000 pg/mL |
| Sensitivity: | 9.38 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human PKM2 in samples. No significant cross-reactivity or interference between Human PKM2 and analogues was observed. |
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 Human PKM2. 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 Human PKM2 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 Human PKM2, 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 Human PKM2. The concentration of Human PKM2 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | PKM2: one of three rate-limiting enzymes in glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding one molecule ATP and a pyruvate molecule, which is a central metabolic intermediate that can be used as a building block or oxidized further. Overexpressed in a variety of cancers. Interacts with thyroid hormone, and thus may mediate cellular metabolic effects induced by thyroid hormones. Binds Opa protein, a bacterial outer membrane protein involved in gonococcal adherence to and invasion of human cells, suggesting a role of this protein in bacterial pathogens is. There are several mammalian isozymes of pyruvate kinase encoded by different genes. The L type predominates in liver, and the M type in muscle and brain. Two alternatively spliced human isoforms of PKM2 have been reported. |
| UniProt Protein Details: | Protein type:Carbohydrate Metabolism - glycolysis and gluconeogenesis; Kinase, other; EC 2. 7. 1. 40; Carbohydrate Metabolism - pyruvate; Mitochondrial; Nucleotide Metabolism - purine Chromosomal Location of Human Ortholog: 15q22 Cellular Component: extracellular matrix; mitochondrion; cytoplasm; plasma membrane; nucleus; cytosol; vesicle; cilium Molecular Function:potassium ion binding; protein binding; magnesium ion binding; pyruvate kinase activity; ATP binding Biological Process: glycolysis; carbohydrate metabolic process; programmed cell death; glucose metabolic process; pathogenesis; phosphorylation |
| NCBI Summary: | This gene encodes a protein involved in glycolysis. The encoded protein is a pyruvate kinase that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP and pyruvate. This protein has been shown to interact with thyroid hormone and may mediate cellular metabolic effects induced by thyroid hormones. This protein has been found to bind Opa protein, a bacterial outer membrane protein involved in gonococcal adherence to and invasion of human cells, suggesting a role of this protein in bacterial pathogenesis. Several alternatively spliced transcript variants encoding a few distinct isoforms have been reported. [provided by RefSeq, May 2011] |
| UniProt Code: | P14618 |
| NCBI GenInfo Identifier: | 20178296 |
| NCBI Gene ID: | 5315 |
| NCBI Accession: | P14618. 4 |
| UniProt Secondary Accession: | P14618,P14786, Q53GK4, Q96E76, Q9BWB5, Q9UCV6, Q9UPF2 A6NFK3, B2R5N8, B3KRY0, B4DFX8, B4DUU6, |
| UniProt Related Accession: | P14618 |
| Molecular Weight: | |
| NCBI Full Name: | Pyruvate kinase PKM |
| NCBI Synonym Full Names: | pyruvate kinase, muscle |
| NCBI Official Symbol: | PKM |
| NCBI Official Synonym Symbols: | PK3; TCB; OIP3; PKM2; CTHBP; THBP1; HEL-S-30 |
| NCBI Protein Information: | pyruvate kinase PKM; p58; OIP-3; tumor M2-PK; PK, muscle type; pyruvate kinase 2/3; OPA-interacting protein 3; pyruvate kinase isozymes M1/M2; pyruvate kinase muscle isozyme; thyroid hormone-binding protein 1; epididymis secretory protein Li 30; cytosolic thyroid hormone-binding protein; thyroid hormone-binding protein, cytosolic |
| UniProt Protein Name: | Pyruvate kinase PKM |
| UniProt Synonym Protein Names: | Cytosolic thyroid hormone-binding protein; CTHBP; Opa-interacting protein 3; OIP-3; Pyruvate kinase 2/3; Pyruvate kinase muscle isozyme; Thyroid hormone-binding protein 1; THBP1; Tumor M2-PK; p58 |
| Protein Family: | Pyruvate kinase |
| UniProt Gene Name: | PKM |
| UniProt Entry Name: | KPYM_HUMAN |
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 |
| 1000 | 51605 55745 | 53675 | 53650 |
| 500 | 20983 23995 | 22489 | 22464 |
| 250 | 10933 9489 | 10211 | 10186 |
| 125 | 4613 5189 | 4901 | 4876 |
| 62.5 | 2595 2311 | 2453 | 2428 |
| 31.25 | 1305 1257 | 1281 | 1256 |
| 15.63 | 684 732 | 708 | 683 |
| 0 | 25 25 | 25 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human PKM2 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PKM2 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 55.31 | 145.07 | 456.58 | 57.29 | 131.22 | 416.35 |
| Standard deviation | 5.76 | 14.72 | 35.11 | 5.63 | 9.25 | 28.56 |
| C V (%) | 10.41 | 10.15 | 7.69 | 9.83 | 7.05 | 6.86 |
Recovery
The recovery of Human PKM2 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) | 84-97 | 90 |
| EDTA plasma (n=5) | 92-107 | 99 |
| Cell culture media (n=5) | 88-101 | 96 |
Linearity
Samples were spiked with high concentrations of Human PKM2 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) | ||
| 1:2 | Range (%) | 99-115 | 88-102 | 88-102 |
| Average (%) | 106 | 94 | 95 | |
| 1:4 | Range (%) | 92-110 | 89-101 | 91-106 |
| Average (%) | 100 | 95 | 97 | |
| 1:8 | Range (%) | 97-112 | 90-102 | 104-116 |
| Average (%) | 104 | 95 | 109 | |
| 1:16 | Range (%) | 89-101 | 97-112 | 86-100 |
| Average (%) | 96 | 105 | 93 |
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 ×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 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 |
- 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 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.
- Determine the RLU value of each well immediately.
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