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PLGF Kit ELISA

PGF Reactivité: Souris Colorimetric Sandwich ELISA 7.8-500 pg/mL Cell Culture Supernatant, Plasma, Serum
N° du produit ABIN456164
  • Antigène Voir toutes PLGF (PGF) Kits ELISA
    PLGF (PGF) (Placenta Growth Factor (PGF))
    Reactivité
    • 6
    • 5
    • 4
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Souris
    Méthode de détection
    Colorimetric
    Type de méthode
    Sandwich ELISA
    Gamme de detection
    7.8-500 pg/mL
    Seuil minimal de détection
    7.8 pg/mL
    Application
    ELISA
    Fonction
    This immunoassay kit allows for the in vitro quantitative determination of mouse PLGF concentrations in cell culture supernates, serum, plasma and other biological fluids.
    Type d'échantillon
    Cell Culture Supernatant, Plasma, Serum
    Analytical Method
    Quantitative
    Specificité
    This assay recognizes recombinant and natural mouse PLGF.
    Réactivité croisée (Details)
    No significant cross-reactivity or interference was observed.
    Sensibilité
    < 1 pg/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.
    Attributs du produit
    Mus musculus,Mouse,Placenta growth factor,PlGF,Pgf,Plgf
    Ingrédients
    Reagent (Quantity): Assay plate (1×20ml), Standard (2), Sample Diluent (1×20ml), Assay Diluent A (1×10ml), Assay Diluent B (1×10ml), Detection Reagent A (1×120 μl), Detection Reagent B (1×120 μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), Instructions 1
    Matériel non inclus
    Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.
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  • Volume d'échantillon
    100 μL
    Plaque
    Pre-coated
    Protocole
    The microtiter plate provided in this kit has been pre-coated with an antibody specific to PLGF. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for PLGF and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain PLGF, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a 2 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 PLGF in the samples is then determined by comparing the O.D. of the samples to the standard curve.
    Préparation des réactifs

    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 pg/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (200 pg/mL). The Sample Diluent serves as the zero standard (0 pg/mL). Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.

    Prélèvement de l'échantillon
    Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20 C or -80 C . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8 C within 30 minutes of collection. Store samples at -20 C or -80 C . Avoid repeated freeze-thaw cycles. Cell culture supernates and 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: Serum, plasma, and cell culture supernatant samples to be used within 7 days may be stored at 2-8 C , otherwise samples must stored at -20 C ( ≤ 1 months) or -80 C ( ≤ 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.
    Procédure de l'essai

    Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
    1. Add 100 μ l 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 μ l of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37C. 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 (approximately 400 μ l) 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, 4 remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    5. Add 100 μ l 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 μ l of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37C. Protect from light.
    8. Add 50 μ l 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. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
    2. 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. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10 μ l for once pipetting.
    3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
    4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
    5. 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.
    6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
    7. Duplication of all standards and specimens, although not required, is recommended.
    8. Substrate Solution is easily contaminated. Please protect it from light.

    Calcul des résultats

    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 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 x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the PLGF concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. 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
  • Conseil sur la manipulation
    1. The kit should not be used beyond the expiration date on the kit label. 3
    2. Do not mix or substitute reagents with those from other lots or sources.
    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.
    Stock
    4 °C/-20 °C
    Stockage commentaire
    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.
  • Antigène Voir toutes PLGF (PGF) Kits ELISA
    PLGF (PGF) (Placenta Growth Factor (PGF))
    Autre désignation
    Pgf (PGF Produits)
    Synonymes
    D12S1900 Kit ELISA, PGFL Kit ELISA, PLGF Kit ELISA, PlGF-2 Kit ELISA, SHGC-10760 Kit ELISA, AI854365 Kit ELISA, PIGF Kit ELISA, Plgf Kit ELISA, PlGF Kit ELISA, placental growth factor Kit ELISA, placenta growth factor Kit ELISA, PGF Kit ELISA, Pgf Kit ELISA, PLGF Kit ELISA
    Sujet
    Based on sequence and structural similarities, Placenta Growth Factor (PlGF) is a member of the Vascular Endothelial Growth Factor (VEGF) family that also includes VEGF/VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E (viral). PlGF is also related, albeit more distantly, to the PDGF family of growth factors. The VEGF family is widely known for its roles in the development and/or growth of the vascular or lymphatic endothelia. The PlGF sequence predicts a 149 amino acid (aa) mature protein with a 21 aa signal sequence and a centrally located PDGF-like domain with 8 conserved cysteine residues that form a cysteine knot structure. PlGF shares approximately 42% aa sequence identity with VEGF, and the two share significant structural similarity. Although PlGF does not share the pro-angiogenic receptor VEGF R2 with VEGF, both bind VEGF R1 (soluble and transmembrane forms), Neuropilin-1, and Neuropilin-2. VEGF and PlGF appear to have different effects on VEGF R1 activity and, subsequently, affect the expression of different downstream genes. PlGF exists in at least 4 alternatively spliced forms: PlGF-1, PlGF-2, PlGF-3, and PlGF-4. Notable differences between these forms include the insertion of a heparin-binding domain in PlGF-2 and PlGF-4 that might result in increased association with the cell membrane or altered affinities for PlGF receptors. In mice, only the PlGF-2 ortholog has been described and shares 65% aa identity with its human counterpart. As the name reflects, PlGF was first identified in human placenta, and indeed, is expressed prominently in placenta under normal conditions. Other tissues and cell types expressing PlGF include microvascular and human umbilical vein endothelia, bone marrow, uterine natural killer cells, and keratinocytes. It is also upregulated under certain pathological conditions including wound healing and tumor formation.
    Pathways
    VEGFR1 Specific Signals
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