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Phosphofructokinase (PFK) Activity Assay Kit
SKU: E-BC-K612-M-96
Phosphofructokinase (PFK) Activity Assay Kit
| SKU # | E-BC-K612-M |
| Detection Instrument | Microplate reader (330-350 nm, optimum wavelength: 340 nm) |
| Detection method | Colorimetric method |
Product Details
Properties
| Synonyms | PFK |
| Sample type | Serum, plasma, tissue and cell sample |
| Sensitivity | 0.27 U/L |
| Detection range | 0.27-32.29 U/L |
| Detection Method | Colorimetric method |
| Assay type | Enzyme Activity |
| Assay time | 15 min |
| Precision | Average inter-assay CV: 8% | Average intra-assay CV: 3% |
| Other instruments required | Centrifuge |
| Other reagents required |
Normal saline (0.9% NaCl) |
| Storage | -20℃ |
| Valid period | 12 months |
Dilution of Sample
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| 10% Rat liver tissue homogenate | 1 |
| 10% Rat kidney tissue homogenate | 1 |
| 10% Rat brain tissue homogenate | 1 |
| 10% Mouse liver tissue homogenate | 1 |
| 10% Mouse spleen tissue homogenate | 1 |
| 10% Mouse kidney tissue homogenate | 1 |
| 10% Mouse heart tissue homogenate | 1 |
| Rat serum | 1 |
| Rat plasma | 1 |
| Jurkat cell | 1 |
Note: The diluent is normal saline (0.9% NaCl). For the dilution of other sample types, please do pretest to confirm the dilution factor.
Detection Principle
Phosphofructokinase(also known as 6-phosphofructokinase; PFK) is a class of kinases that act on fructose-6-phosphate. Phosphofructokinase catalyzes fructose-6-phosphate and ATP to produce fructose-1, 6-diphosphate and ADP, and pyruvate kinase and lactate dehydrogenase further catalyze the oxidation of NADH to NAD+. The activity of PFK can be reflected by the determination of NADH decline rate at 340 nm.
Kit Components & Storage
| Item | Component | Size 1(48 T) | Size 2(96 T) | Storage |
| Reagent 1 | Buffer Solution | 10 mL ×1 vial | 20 mL × 1 vial | -20℃, 12 months, shading light |
| Reagent 2 | Substrate A | Powder × 1 vial | Powder × 2 vials | -20°C, 12 months shading light |
| Reagent 3 | Substrate B | Powder × 1 vial | Powder × 2 vials | -20°C, 12 months shading light |
| Reagent 4 | Enzyme Reagent | Powder × 1 vial | Powder × 2 vials | -20°C, 12 months shading light |
| UV Microplate | 96 wells | No requirement | ||
| Plate Sealer | 2 pieces | |||
Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.
Technical Data:
Parameter:
Intra-assay Precision
Three human serum samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U/L) | 2.60 | 18.40 | 26.50 |
| %CV | 3.5 | 3.1 | 2.4 |
Inter-assay Precision
Three human serum samples were assayed 20 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U/L) | 2.60 | 18.40 | 26.50 |
| %CV | 7.6 | 8.5 | 7.9 |
Recovery
Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 100%
| Sample 1 | Sample 2 | Sample 3 | |
| Expected Conc. (U/L) | 8.5 | 22.3 | 29.5 |
| Observed Conc. (U/L) | 8.4 | 22.7 | 29.2 |
| Recovery rate (%) | 99 | 102 | 99 |
Sensitivity
The analytical sensitivity of the assay is 0.27 U/L. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.