Mouse Metabolism ELISA Kits
Mouse cPLA2 (Phospholipase A2, Cytosolic) CLIA Kit (MOES00612)
- SKU:
- MOES00612
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Mouse
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Metabolism
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Mouse |
| Detection method: | Chemiluminescence |
| Detection range: | 31.25-2000 pg/mL |
| Sensitivity: | 18.75 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Mouse cPLA2 in samples. No significant cross-reactivity or interference between Mouse cPLA2 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 Mouse cPLA2. 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 cPLA2 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 cPLA2, 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 cPLA2. The concentration of Mouse cPLA2 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | cPLA2: a calcium-dependent phospholipase A2 that catalyzes the release of arachidonic acid from membrane phospholipids. Selectively hydrolyzes arachidonyl phospholipids in the sn-2 position releasing arachidonic acid. Arachidonic acid is a precursor for the eicosanoids that are involved in hemodynamic regulation, inflammatory responses, and other cellular processes. Promotes cerebellar long-term depression and motor learning. Translocates to the Golgi and endoplasmic reticulum in a calcium-dependent fashion. Translocation and activation of at the ER facilitates the process of ER stress. Regulates the biogenesis of lipid droplets, a process dependent on JNK and ceramide kinase. Stimulated by agonists such as ATP, EGF, thrombin and bradykinin as well as by cytosolic Ca2+. The N-terminal C2 domain, by its association with lipid membranes, mediates the regulation of CPLA2 by presenting the active site to its substrate in response to elevations of cytosolic Ca2+. Inhibited when in trimolecular complex with ANXA2 and S100A10. Phosphorylation of S727 relieves this inhibitory interaction, thus activating PLA2G4A. |
| UniProt Protein Details: | Protein type:Lipid Metabolism - linoleic acid; Lipid Metabolism - arachidonic acid; Lipid Metabolism - ether lipid; Lipid Metabolism - alpha-linolenic acid; Phospholipase; EC 3. 1. 1. 4; Lipid Metabolism - glycerophospholipid; EC 3. 1. 1. 5 Cellular Component: cytoplasm; cytoplasmic vesicle; cytosol; endoplasmic reticulum; Golgi apparatus; intracellular membrane-bound organelle; nucleus; perinuclear region of cytoplasm Molecular Function:calcium ion binding; calcium-dependent phospholipase A2 activity; calcium-dependent phospholipid binding; histone acetyltransferase binding; hydrolase activity; lysophospholipase activity; metal ion binding; phospholipase A2 activity; phospholipase activity Biological Process: arachidonic acid metabolic process; arachidonic acid secretion; decidualization; icosanoid biosynthetic process; lipid catabolic process; lipid metabolic process; metabolic process; ovulation from ovarian follicle; phospholipid catabolic process; positive regulation of apoptosis; positive regulation of cell proliferation; positive regulation of inflammatory response; positive regulation of prostaglandin biosynthetic process; positive regulation of vesicle fusion; regulation of cell proliferation; response to calcium ion; response to organic substance; surfactant homeostasis |
| NCBI Summary: | The protein encoded by this gene is a member of the phospholipase A2 group IV family. This enzyme hydrolyzes membrane phospholipids, thereby releasing the polyunsaturated fatty acid, arachidonic acid. Arachidonic acid is further metabolized into eicosanoids such as leukotrienes, thromboxanes and prostaglandins, that play important roles in regulating diverse biological processes such as inflammatory responses, membrane and actin dynamics, and tumorigenesis. A rise in intracellular calcium levels results in binding of calcium to the C2 domain of this protein, and triggers the translocation from the cytosol to intracellular membranes, including the Golgi apparatus. Disruption of this gene in mice led to decreased levels of eicosonaoids and platelet-activating factor, decreased allergic symptoms, and impaired reproductive ability in females. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Mar 2015] |
| UniProt Code: | P47713 |
| NCBI GenInfo Identifier: | 6679369 |
| NCBI Gene ID: | 18783 |
| NCBI Accession: | NP_032895. 1 |
| UniProt Related Accession: | P47713 |
| Molecular Weight: | 85,222 Da |
| NCBI Full Name: | cytosolic phospholipase A2 isoform 1 |
| NCBI Synonym Full Names: | phospholipase A2, group IVA (cytosolic, calcium-dependent) |
| NCBI Official Symbol: | Pla2g4a |
| NCBI Official Synonym Symbols: | cPLA2; Pla2g4; cPLA2alpha; cPLA2-alpha |
| NCBI Protein Information: | cytosolic phospholipase A2 |
| UniProt Protein Name: | Cytosolic phospholipase A2 |
| UniProt Synonym Protein Names: | Phospholipase A2 group IVAIncluding the following 2 domains:Phospholipase A2 (EC:3. 1. 1. 4)Alternative name(s):Phosphatidylcholine 2-acylhydrolase |
| Protein Family: | Cytosolic phospholipase |
| UniProt Gene Name: | Pla2g4a |
| UniProt Entry Name: | PA24A_MOUSE |
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 |
| 2000 | 53025 58417 | 55721 | 55693 |
| 1000 | 21702 26246 | 23974 | 23946 |
| 500 | 11196 10868 | 11032 | 11004 |
| 250 | 5111 5477 | 5294 | 5266 |
| 125 | 2816 2400 | 2608 | 2580 |
| 62.5 | 1341 1281 | 1311 | 1283 |
| 31.25 | 632 716 | 674 | 646 |
| 0 | 27 29 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse cPLA2 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse cPLA2 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) | 107.39 | 238.21 | 829.58 | 107.72 | 246.78 | 823.30 |
| Standard deviation | 12.41 | 22.73 | 55.50 | 11.35 | 26.01 | 68.09 |
| C V (%) | 11.56 | 9.54 | 6.69 | 10.54 | 10.54 | 8.27 |
Recovery
The recovery of Mouse cPLA2 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) | 88-101 | 93 |
| EDTA plasma (n=5) | 98-113 | 106 |
| Cell culture media (n=5) | 97-114 | 105 |
Linearity
Samples were spiked with high concentrations of Mouse cPLA2 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 (%) | 85-99 | 90-103 | 92-107 |
| Average (%) | 91 | 96 | 99 | |
| 1:4 | Range (%) | 95-108 | 101-116 | 95-111 |
| Average (%) | 101 | 109 | 103 | |
| 1:8 | Range (%) | 86-100 | 95-111 | 100-116 |
| Average (%) | 92 | 102 | 107 | |
| 1:16 | Range (%) | 91-106 | 90-103 | 92-106 |
| Average (%) | 97 | 97 | 100 |
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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