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Apelin Kit ELISA

APLN Reactivité: Humain, Rat, Souris Colorimetric Competition ELISA 0.1-1.000 ng/mL Cell Culture Supernatant, Serum
N° du produit ABIN1979332
  • Antigène Voir toutes Apelin (APLN) Kits ELISA
    Apelin (APLN)
    Reactivité
    • 4
    • 3
    • 2
    • 1
    • 1
    • 1
    Humain, Rat, Souris
    Méthode de détection
    Colorimetric
    Type de méthode
    Competition ELISA
    Gamme de detection
    0.1-1.000 ng/mL
    Seuil minimal de détection
    0.1 ng/mL
    Application
    ELISA
    Fonction
    Human/Mouse/Rat Apelin EIA Kit optimized for serum and cell culture medium. Competition-based ELISA on a 96-well strip plate.
    Type d'échantillon
    Cell Culture Supernatant, Serum
    Analytical Method
    Quantitative
    Specificité
    This kit is designed to target the C-terminus of the 77-aa apelin peptide and therefore is expected to detect all active forms of Apelin including Apelin-36, Apelin-31, Apelin-28 and Apelin 13 can be detected.

    Cross Reactivity: This kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, and NPY.
    Réactivité croisée (Details)
    This kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, and NPY. This kit is designed to target the C-terminus of the 77-aa apelin peptide and therefore is expected to detect all active forms of Apelin including Apelin-36, Apelin-31, Apelin-28 and Apelin 13 can be detected.
    Sensibilité
    5.84 pg/mL
    Attributs du produit
    • Strip plates and additional reagents allow for use in multiple experiments
    • Quantitative protein detection
    • Establishes normal range
    • The best products for confirmation of antibody array data
    Ingrédients
    • Pre-Coated 96-well Strip Microplate
    • Wash Buffer
    • Standard Peptide
    • Assay Diluent(s)
    • Biotinylated Peptide
    • HRP-Streptavidin
    • TMB One-Step Substrate
    • Stop Solution
    • Assay Diagram
    • Positive Control Sample
    • Capture Antibody
    • User Manual
    Matériel non inclus
    • Distilled or deionized water
    • Precision pipettes to deliver 2 μL to 1 mL volumes
    • Adjustable 1-25 mL pipettes for reagent preparation
    • 100 mL and 1 liter graduated cylinders
    • Tubes to prepare standard and sample dilutions
    • Orbital shaker
    • Aluminum foil
    • Saran Wrap
    • Absorbent paper
    • Microplate reader capable of measuring absorbance at 450nm
    • SigmaPlot software (or other software that can perform four-parameter logistic regression models)
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  • Indications d'application
    Recommended Dilution for serum and plasma samplesHuman: 2X / Mouse: 2X / Rat: 2X
    Volume d'échantillon
    100 μL
    Durée du test
    5 h
    Plaque
    Pre-coated
    Protocole
    1. Prepare all reagents, samples and standards as instructed.
    2. Add 100 μL detection antibody to each well.
    3. Incubate 1.5 h at RT or O/N at 4 °C.
    4. Add 100 μL standard or sample to each well.
    5. Incubate 2.5 h at RT.
    6. Add 100 μL prepared streptavidin solution.
    7. Incubate 45 min at RT.
    8. Add 100 μL TMB One-Step Substrate Reagent to each well.
    9. Incubate 30 min at RT.
    10. Add 50 μL Stop Solution to each well.
    11. Read plate at 450 nm immediately.
    Préparation des réactifs
    1. Keep kit reagents on ice during steps. Equilibrate plate to room temperature before opening the sealed pouch.
      2. Briefly centrifuge the APC Antibody vial (Item N) and reconstitute with 5 µL of ddH2O before use. Add 50 µL of 1x Assay Diluent E into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently.
      3. The antibody concentrate should then be diluted 100-fold with 1x Assay Diluent E. This is your anti-Apelin C-Terminus antibody working solution, which will be used in step 2 of the Assay Procedure. NOTE: the following steps may be done during the antibody incubation procedure (step 2 of Assay Procedure).
      4. Briefly centrifuge the vial of biotinylated Apelin peptide (Item F) and reconstitute with 20 µL of ddH2O before use. Add 5 µL of Item F to 5 mL 1X Assay Diluent E. Pipette up and down to mix gently. The final concentration of biotinylated Apelin C- Terminus will be 50 ng/mL. This solution will only be used as the diluent in step 5 of Reagent Preparation.
      5. Preparation of Standards: Label 6 microtubes with the following concentrations: 1000 ng/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 100 pg/mL and 0 pg/mL. Pipette 450 mL of biotinylated Apelin C-Terminus solution into each tube, except for the 1000 ng/mL (leave this one empty). It is very important to make sure the concentration of biotinylated Apelin C-Terminus is 50 ng/mL in all standards. a. Briefly centrifuge the vial of standard Apelin peptide (Item C) and reconstitute with 10 µL of ddH2O. In the tube labeled 1000 ng/mL, pipette 8 µL of Item C and 792 µL of 50 ng/mL biotinylated Apelin C-Terminus solution (prepared in step 4 above). This is your Apelin C-Terminus stock solution (1000 ng/mL Apelin C-Terminus, 50 ng/mL biotinylated Apelin C- Terminus). Mix thoroughly. This solution serves as the first standard. b. To make the 100 ng/mL standard, pipette 50 µL of Apelin C- Terminus stock solution into the tube labeled 100 ng/mL. Mix thoroughly. c. Repeat this step with each successive concentration, preparing a dilution series as shown in the illustration below. Each time, use 450 mL of biotinylated Apelin C-Terminus and 50 mL of the prior concentration until 100 pg/mL is reached. Mix each tube thoroughly before the next transfer. d. The final tube (0 pg/mL Apelin C-Terminus, 50 ng/mL biotinylated Apelin C-Terminus) serves as the zero standard (or total binding).
      6. Prepare a 10-fold dilution of Item F. To do this, add 2 mL of Item F to 18 mL of the 1X Assay Diluent E. This solution will be used in steps 7 and
      9.
      7. Positive Control Preparation: Briefly centrifuge the positive control vial and reconstitute with 100 µL of ddH2O before use (Item M). To the tube of Item M, add 101 µL 1x Assay Diluent E. Also add 2 µL of 10-fold diluted Item F (prepared in step 6) to the tube. This is a 2-fold dilution of the positive control. Mix thoroughly. The positive control is a cell culture medium sample that is meant to be a system control (to verify that the detection & kit components are working). It may be diluted further if desired, but be sure the final concentration of biotinylated Apelin C-Terminus is 50 ng/mL.
      8. If Item B (20X Wash Concentrate) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to yield 400 mL of 1X Wash Buffer.
      9. Sample Preparation: Use 1X Assay Diluent E + biotinylated APC to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Apelin C-Terminus is 50 ng/mL in every sample.
      Example: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Apelin C-Terminus to a final concentration of 50 ng/mL.
      Example: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.
      10. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. The HRP-Streptavidin concentrate should be diluted 100- fold with 1X Assay Diluent E.
    Préparation de l'échantillon

    Use 1X Assay Diluent E + biotinylated APC to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Apelin C-Terminus is 50 ng/mL in every sample. EXAMPLE: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Apelin C-Terminus to a final concentration of 50 ng/mL. EXAMPLE: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.

    Procédure de l'essai
    1. Keep kit reagents on ice during reagent preparation steps. It is recommended that all standards and samples be run at least in duplicate.
      2. Add 100 µL Anti-Apelin C-Terminus antibody (see Reagent Preparation step 3) to each well. Incubate for 1.5 hours at room temperature with gentle shaking (1-2 cycles/sec). You may also incubate overnight at 4 °C. 0
      3. Discard the solution and wash wells 4 times with 1X Wash Buffer (200-300 µL each). Washing may be done with a multichannel pipette or an automated plate washer. Complete removal of liquid at each step is essential to good assay performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
      4. Add 100 µL of each standard (see Reagent Preparation step 5), positive control (see Reagent Preparation step 7) and sample (see Reagent Preparation step 9) into appropriate wells. Be sure to include a blank well (Assay Diluent only). Cover wells and incubate for 2.5 hours at room temperature with gentle shaking (1-2 cycles/sec) or overnight at 4 °C.
      5. Discard the solution and wash 4 times as directed in Step
      3.
      6. Add 100 µL of prepared HRP-Streptavidin solution (see Reagent Preparation step 10) to each well. Incubate for 45 minutes at room temperature with gentle shaking. It is recommended that incubation time should not be shorter or longer than 45 minutes.
      7. Discard the solution and wash 4 times as directed in Step
      3.
      8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking (1-2 cycles/sec).
      9. Add 50 µL of Stop Solution (Item I) to each well. Read absorbances at 450 nm immediately. 1
    Calcul des résultats

    Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the blank optical density. Plot the standard curve using SigmaPlot software (or other software which can perform four-parameter logistic regression models), with standard concentration on the x-axis and percentage of absorbance on the y-axis. Draw the best-fit curve through the standard points.

    Précision du teste
    Intra-Assay: CV < 10 %
    Inter-Assay: CV < 15 %
    Restrictions
    For Research Use only
  • Conseil sur la manipulation
    Avoid repeated freeze/thaw cycles.
    Stock
    -20 °C
    Stockage commentaire
    Standard, Biotinylated Apelin C-Terminus peptide, and Positive Control should be stored at -20°C after arrival. Avoid multiple freeze-thaws. The remaining kit components may be stored at 4°C. Opened Microplate Wells and antibody (Item N) may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.
    Date de péremption
    6 months
  • Principe, Melgar-Lesmes, Fernández-Varo, del Arbol, Ros, Morales-Ruiz, Bernardi, Arroyo, Jiménez: "The hepatic apelin system: a new therapeutic target for liver disease." dans: Hepatology (Baltimore, Md.), Vol. 48, Issue 4, pp. 1193-201, (2008) (PubMed).

    Lee, George, ODowd: "Unravelling the roles of the apelin system: prospective therapeutic applications in heart failure and obesity." dans: Trends in pharmacological sciences, Vol. 27, Issue 4, pp. 190-4, (2006) (PubMed).

    Lee, Lança, Cheng, Nguyen, Ji, Gobeil, Chemtob, George, ODowd: "Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors." dans: The Journal of biological chemistry, Vol. 279, Issue 9, pp. 7901-8, (2004) (PubMed).

    Lee, Saldivia, Nguyen, Cheng, George, ODowd: "Modification of the terminal residue of apelin-13 antagonizes its hypotensive action." dans: Endocrinology, Vol. 146, Issue 1, pp. 231-6, (2004) (PubMed).

    Lee, Cheng, Nguyen, Fan, Kariyawasam, Liu, Osmond, George, ODowd: "Characterization of apelin, the ligand for the APJ receptor." dans: Journal of neurochemistry, Vol. 74, Issue 1, pp. 34-41, (2000) (PubMed).

  • Antigène Voir toutes Apelin (APLN) Kits ELISA
    Apelin (APLN)
    Autre désignation
    Apelin (APLN Produits)
    Synonymes
    APLN Kit ELISA, Xapelin Kit ELISA, xnpep2 Kit ELISA, preproapelin Kit ELISA, 6030430G11Rik Kit ELISA, Apel Kit ELISA, APEL Kit ELISA, XNPEP2 Kit ELISA, apelin Kit ELISA, apelin S homeolog Kit ELISA, apln Kit ELISA, APLN Kit ELISA, apln.S Kit ELISA, Apln Kit ELISA
    Sujet
    Apelin, an endogenous ligand for the G-protein-coupled APJ receptor, has been recently extensively studied in obesity research. It is not only expressed in adipocyte tissue, but also widely expressed in various other organs such as the heart, lung, kidney, gastrointestinal tract, brain, adrenal glands, endothelium, and human plasma. Apelin is derived from a 77-amino-acid prepropeptide that is cleaved into a 55-amino-acid fragment and then into shorter forms. The physiologically active form is thought to be apelin 36, although the pyroglutamylated form of apelin 13, which is also produced endogenously, is more potent. Studies have shown the association between apelin and obesity. Apelin has higher circulating levels in obesity. Insulin exerts a positive action on adipocyte apelin production. Apelin also regulates fluid homeostasis, playing an important role in the hypothalamic regulation of food and water intake, and pituitary hormone release. In addition to its role in obesity, apelin acts as a mediator of cardiovascular control, including for blood pressure and blood flow. It is one of the most potent stimulators of cardiac contractility yet identified, and plays a role in cardiac tissue remodeling. Apelin levels are increased in left ventricles of patients with chronic heart failure and also in patients with chronic liver disease.
    ID gène
    30878
    UniProt
    Q9R0R4
    Pathways
    Positive Regulation of Peptide Hormone Secretion, Hormone Activity, Feeding Behaviour
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