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PLA2G4A ELISA Kit

PLA2G4A Reactivity: Human Colorimetric Sandwich ELISA 3.12-200 U/mL Cell Lysate, Tissue Homogenate
Catalog No. ABIN454933
  • Target See all PLA2G4A ELISA Kits
    PLA2G4A (Phospholipase A2, Group IVA (Cytosolic, Calcium-Dependent) (PLA2G4A))
    Reactivity
    • 4
    • 4
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Human
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Detection Range
    3.12-200 U/mL
    Minimum Detection Limit
    3.12 U/mL
    Application
    ELISA
    Purpose
    This immunoassay kit allows for the use in vitro quantitative determination of human cytosolic phospholipase A2, cPLA2 concentrations in tissue homogenates, cell lysates and other biological fluids.
    Sample Type
    Cell Lysate, Tissue Homogenate
    Analytical Method
    Quantitative
    Specificity
    This assay recognizes recombinant and natural human cPLA2.
    Cross-Reactivity (Details)
    No significant cross-reactivity or interference was observed.
    Sensitivity
    < 0.78 U/mL
    The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.
    Characteristics
    Homo sapiens,Human,Cytosolic phospholipase A2,cPLA2,Phospholipase A2 group IVA,PLA2G4A,CPLA2,PLA2G4
    Components
    Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay DiluentB 1 x 10ml Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml), Plate sealer for 96 wells (1x5)
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  • Sample Volume
    100 μL
    Plate
    Pre-coated
    Protocol
    The microtiter plate provided in this kit has been pre-coated with an antibody specific to cPLA2. 2 Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for cPLA2 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain cPLA2, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of cPLA2 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
    Reagent Preparation

    Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 200 U/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (200 U/mL). The Sample Diluent serves as the zero standard (0 U/mL). U/mL 200 100 50 25 12.5 6.25 3.12 0 Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

    Sample Collection
    Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20 °C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20 °C. Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles. Note: Tissue homogenates and cell lysates samples to be used within 7 days may be stored at 2-8C, otherwise samples must stored at -20 °C (≤ 3 months) or -80 °C (≤ 6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. It is recommended that all samples be assayed in duplicate.
    Assay Procedure

    Allow all reagents to reach room temperature. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Arrange and label required number of strips. Prepare all reagents, working standards and samples as directed in the previous sections.
    1. Add 100 uL of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 °C.
    2. Remove the liquid of each well, don’t wash.
    3. Add 100 uL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37°C. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
    4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by 4 aspirating or decanting. Invert the plate and blot it against clean paper towels.
    5. Add 100 uL of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 °C.
    6. Repeat the aspiration/wash as in step
    4. 7. Add 90 uL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37°C. Protect from light.
    8. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
    9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
    Important Note:
    1. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once.
    2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
    3. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
    4. Duplication of all standards and specimens, although not required, is recommended.
    5. When mixing or reconstituting protein solutions, always avoid foaming.
    6. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
    7. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
    8. Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.

    Calculation of Results

    Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer 5 software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the cPLA2 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

    Restrictions
    For Research Use only
  • Handling Advice
    1. The kit should not be used beyond the expiration date on the kit label.
    2. Do not mix or substitute reagents with those from other lots or sources. 3
    3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
    4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
    Storage
    4 °C/-20 °C
    Storage Comment
    The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.
  • Target See all PLA2G4A ELISA Kits
    PLA2G4A (Phospholipase A2, Group IVA (Cytosolic, Calcium-Dependent) (PLA2G4A))
    Alternative Name
    PLA2G4A (PLA2G4A Products)
    Synonyms
    PLA2G4 ELISA Kit, cPLA2-alpha ELISA Kit, Pla2c ELISA Kit, Pla2g4 ELISA Kit, cPLA2 ELISA Kit, pla2g4 ELISA Kit, CPLA2 ELISA Kit, PLA2G4A ELISA Kit, PLA2 ELISA Kit, PLA2G1B ELISA Kit, cPLA2alpha ELISA Kit, pla2g4a ELISA Kit, si:dkey-97o5.1 ELISA Kit, phospholipase A2 group IVA ELISA Kit, phospholipase A2 group IVA L homeolog ELISA Kit, phospholipase A2, group IVA (cytosolic, calcium-dependent) ELISA Kit, phospholipase A2, group IVAb (cytosolic, calcium-dependent) ELISA Kit, PLA2G4A ELISA Kit, Pla2g4a ELISA Kit, pla2g4a.L ELISA Kit, pla2g4ab ELISA Kit
    Background
    Phospholipases A2 (PLA2s) EC 3.1.1.4 are enzymes that specifically hydrolyze the sn-2 fatty acid acyl bond of phospholipids, producing a free fatty acid and a lyso-phospholipid. A large range of fatty acids can be bound to the sn2 position of phospholipids, including arachidonic acid and eicosapentaenoic acid. Phospholipases A2 include several unrelated protein families with common enzymatic activity. Two most notable families are secreted and cytosolic phospholipases A2. Other families include Ca2+ independent PLA2 (iPLA2) and lipoprotein-associated PLA2s (lp-PLA2), also known as platelet activating factor acetylhydrolase (PAF-AH). Secreted phospholipases A2 (sPLA2): The extracellular forms of phospholipases A2 have been isolated from different venoms (snake, bee, and wasp), from virtually every studied mammalian tissue (including pancreas and kidney) as well as from bacteria. They require Ca2+ for activity. Pancreatic PLA2 serve for the initial digestion of phospholipid compounds in dietary fat. Venom phospholipases help to immobilize prey by promoting cell lysis. Cytosolic phospholipases A2 (cPLA2): The intracellular PLA2 are also Ca-dependent, but they have completely different 3D structure and significantly larger than secreted PLA2 (more than 700 residues). They include C2 domain and large catalytic domain. These phospholipases are involved in cell signaling processes, such as inflammatory response. The produced Arachidonic acid is both a signaling molecule and the precursor for other signalling molecules termed eicosanoids. These include leukotrienes and prostaglandins. Some eicosanoids are synthesized from diacylglycerol, released from the lipid bilayer by phospholipase C (see below). Phospholipases A2 can be classified based on sequence homology.
    Pathways
    Inositol Metabolic Process, G-protein mediated Events, VEGF Signaling
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