Mouse iPLA2 / Phospholipase A2, Calcium Independent ELISA Kit

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SKU:
MOFI00947
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P97819
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
iPLA2, Phospholipidase A2, Calcium Independent, PLA2G6, CaI-PLA2, GVI, INAD1, IPLA2-VIA, NBIA2, NBIA2A, NBIA2B, PARK14, PLA2, PNPLA9, GVIPLA2, iPLA2beta, Patatin-like phospholipase domain-containing protein 9, Group VI phospholipase A2, Intracellular
Reactivity:
Mouse
Frequently bought together:

Description

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Product Name:Mouse iPLA2 / Phospholipase A2, Calcium Independent ELISA Kit
Product Code:MOFI00947
Size:96 Assays
Alias:iPLA2, Phospholipidase A2, Calcium Independent, PLA2G6, CaI-PLA2, GVI, INAD1, IPLA2-VIA, NBIA2, NBIA2A, NBIA2B, PARK14, PLA2, PNPLA9, GVIPLA2, iPLA2beta, Patatin-like phospholipase domain-containing protein 9, Group VI phospholipase A2, Intracellular membrane-associated calcium-independent phospholipase A2 beta
Detection Method:Sandwich ELISA
Application:This immunoassay kit allows for the in vitro quantitative determination of Mouse iPLA2 concentrations in serum plasma and other biological fluids.
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Mouse iPLA2 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse iPLA2 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)85-10296
EDTA plasma(n=5)90-10096
UFH plasma(n=5)87-10293
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse iPLA2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)94-105%89-101%92-104%
EDTA plasma(n=5)89-99%82-97%83-93%
UFH plasma(n=5)81-99%89-99%81-99%
Intra Assay:CV <8%
Inter Assay:CV <10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8-12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotP97819
UniProt Protein Function:PLA2G6: Catalyzes the release of fatty acids from phospholipids. It has been implicated in normal phospholipid remodeling, nitric oxide-induced or vasopressin-induced arachidonic acid release and in leukotriene and prostaglandin production. May participate in fas mediated apoptosis and in regulating transmembrane ion flux in glucose-stimulated B-cells. Has a role in cardiolipin (CL) deacylation. Required for both speed and directionality of monocyte MCP1/CCL2-induced chemotaxis through regulation of F- actin polymerization at the pseudopods. Defects in PLA2G6 are the cause of neurodegeneration with brain iron accumulation type 2B (NBIA2B). A neurodegenerative disorder associated with iron accumulation in the brain, primarily in the basal ganglia. It is characterized by progressive extrapyramidal dysfunction leading to rigidity, dystonia, dysarthria and sensorimotor impairment. Defects in PLA2G6 are the cause of neurodegeneration with brain iron accumulation type 2A (NBIA2A); also known as Seitelberger disease. NBIA2A is a neurodegenerative disease characterized by pathologic axonal swelling and spheroid bodies in the central nervous system. Onset is within the first 2 years of life with death by age 10 years. Defects in PLA2G6 are the cause of Parkinson disease type 14 (PARK14). An adult-onset progressive neurodegenerative disorder characterized by parkinsonism, dystonia, severe cognitive decline, cerebral and cerebellar atrophy and absent iron in the basal ganglia on magnetic resonance imaging. 4 isoforms of the human protein are produced by alternative splicing. Protein type: Lipid Metabolism - ether lipid; Lipid Metabolism - linoleic acid; Lipid Metabolism - alpha-linolenic acid; Lipid Metabolism - glycerophospholipid; EC 3.1.1.4; Phospholipase; Lipid Metabolism - arachidonic acid Cellular Component: mitochondrion; membrane; cytoplasm; microtubule organizing center; cytosol Molecular Function: calmodulin binding; phospholipase A2 activity; ATP-dependent protein binding; calcium-independent phospholipase A2 activity; hydrolase activity; protein kinase binding Biological Process: cardiolipin biosynthetic process; urinary bladder smooth muscle contraction; elevation of cytosolic calcium ion concentration; metabolic process; negative regulation of synaptic transmission, glutamatergic; lipid metabolic process; positive regulation of protein amino acid phosphorylation; chemotaxis; positive regulation of vasodilation; memory; lipid catabolic process; positive regulation of exocytosis
UniProt Protein Details:
NCBI Summary:
UniProt Code:P97819
NCBI GenInfo Identifier:312222739
NCBI Gene ID:53357
NCBI Accession:NP_001185952.1
UniProt Secondary Accession:P97819,Q99LA9, Q9JK61
UniProt Related Accession:P97819
Molecular Weight:28.3kDa
NCBI Full Name:85/88 kDa calcium-independent phospholipase A2 isoform 2
NCBI Synonym Full Names:phospholipase A2, group VI
NCBI Official Symbol:Pla2g6  
NCBI Official Synonym Symbols:iPLA2; PNPLA9; BB112799; iPLA2beta; iPLA(2)beta  
NCBI Protein Information:85/88 kDa calcium-independent phospholipase A2; GVI PLA2; caI-PLA2; iPLA2-beta; group VI phospholipase A2; 85 kDa calcium-independent phospholipase A2; patatin-like phospholipase domain-containing protein 9; intracellular membrane-associated calcium-independent phospholipase A2 beta
UniProt Protein Name:85/88 kDa calcium-independent phospholipase A2
UniProt Synonym Protein Names:Group VI phospholipase A2; GVI PLA2; Intracellular membrane-associated calcium-independent phospholipase A2 beta; iPLA2-beta; Patatin-like phospholipase domain-containing protein 9
Protein Family:
UniProt Gene Name:Pla2g6  
UniProt Entry Name:PLPL9_MOUSE

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4. Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12. Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol
Serum:

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma:

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 - g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid:

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant:

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates:

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.

Tissue homogenates:

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates:

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Breast Milk:

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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