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Urocortin 2 ELISA Kit

UCN2 Reactivity: Mouse Colorimetric Competition ELISA
Catalog No. ABIN956058
  • Target See all Urocortin 2 (UCN2) ELISA Kits
    Urocortin 2 (UCN2)
    Reactivity
    • 3
    • 3
    • 2
    Mouse
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Application
    ELISA
    Analytical Method
    Quantitative
    Specificity
    No cross-reactivity with mouse or rat Urocortin 1, mouse or rat Urocortin 3, mouse, rat or human CRF.
    Characteristics
    Urocortin 2 (Ucn 2), also known as Stresscopin-Related Peptide, is a novel predicted neuropeptide related to corticotropin-releasing factor (CRF). The peptide consists of 38 amino acid residues and was first demonstrated to be expressed centrally and to bind selectively to type 2 CRF receptor (CRFR2). In rodents, Ucn 2 transcripts were shown to be expressed in the discrete regions of the central nervous system including stress-related cell groups in the hypothalamus and brainstem. More recently, the expression of Ucn 2 transcripts was detected in the olfactory bulb, pituitary, cortex, hypothalamus, and spinal cord. Ucn 2 mRNA was also found to be expressed widely in a variety of peripheral tissues, especially in the skin and skeletal muscle tissues. Ucn 2-like immuno-reactivity was detected by RIA in acid extracts of mouse brain, muscle, and skin. Ucn 2 was found by immunohistochemical staining in both skin epidermis and adnexal structures and in the skeletal muscle myocytes. Ucn 2 gene transcription was stimulated in the hypothalamus and brainstem by glucocorticoid administration to the mouse and inhibited by removal of glucocorticoids by adrenal- ectomy, suggesting a putative link between the CRFR1 and CRFR2 pathways. On the other hand, in rats a stressor- specific regulation of Ucn 2 mRNA expression in the hypothalamic paraventricular nucleus was demonstrated, which raised the possibility of a modulary role of Ucn 2 mRNA in stress-induced alteration of anterior and posterior pituitary function, depending on the type of stress. Administration of dexamethasone to mice resulted in a decrease of Ucn 2 mRNA levels in the back skin region. Adrenalectomy signifcantly increased Ucn 2 mRNA levels in the skin, and the levels were reduced back to normal levels after corticoid replacement.
    CRFR2 is found in cardiomyocytes and in endothelial and smooth muscle cells of the systemic vasculature. Ucn 2 is expressed in the mouse cardiomyocytes. In mice, Ucn 2 treatment augmented heart rate, exhibited potent inotropic and lusitropic actions on the left ventricle, and induced a downward shift of the diastolic pressure-volume relation. Ucn 2 also reduced systemic arterial pressure, associated with a lowering of systemic arterial elastance and systemic vascular resistance. The effects of Ucn 2 were specific to CRFR2 function and independent of beta-adrenergic receptors. These experiments demonstrated the potent cardiovascular physiologic actions of Ucn 2 in the both wild-type and cardiomyopathic mice and support a potential beneficial use of Ucn 2 in congestive heart failure treatment. The use of Ucn 2 was also proposed to treat ischemic heart disease because of its potent cardio-protective effect in the mouse heart and its minimal impact on the hypothalamic stress axis. Administration of Ucn 2 to mice prevented the loss of skeletal muscle mass resulting from disuse due to casting, corticosteroid treatment, and nerve damage. In addition, Ucn 2 treatment prevented the loss of skeletal muscle force and myocyte cross-sectional area that accompanied muscle mass losses resulting from disuse due to casting. In normal muscles of the mouse, Ucn 2 increased skeletal muscle mass and force. It was thus proposed that Ucn 2 might find utility in the treatment of skeletal muscle wasting diseases including age-related muscle loss or sarcopenia. Mouse Urocortin 2 (Ucn 2) is a new peptide predicted from mouse cDNA sequence and its physiologic and pathophysiologic significance has not yet been fully elucidated. However, the experimental data presented to date provided evidence for the important physiologic roles of Ucn 2 and urge the necessity of further investigation of the peptide from various points of view. Advantages of this kit include high sensitivity, high specificity, no interference with other sample components. The mouse Urocortin 2 Calibrator is a highly purified synthetic product.This EIA kit for the determination of mouse Urocortin 2 is based on a competitive enzyme immunoassay using a combination of a highly specific antibody to mouse Urocortin 2 and a biotin-avidin affinity system. The 96-well plate is coated with rabbit anti-mouse Urocortin 2 antibody. Mouse Urocortin 2 calibrator or samples and labeled antigen are added to the wells for a competitive immuno-reaction. After incubation and washing, HRP-labeled streptoavidin (SA-HRP) is added to form HRP-labeled streptoavidin-biotinylated mouse Urocortin 2-antibody complexes on the surface of the wells. Finally, HRP enzyme activity is determined by o-Phenylenediamine dihydrochloride (OPD), and the concentration of mouse Urocortin 2 is calculated.
    Components
    1. Antibody-Coated Plate MTP: 1 plate (96-well) Rabbit anti-mouse Urocortin 2 antibody
    2. Urocortin 2 Calibrator Lyophilized: 1 vial (200 ng) Synthetic mouse Urocortin 2
    3. Labeled Antigen Lyophilized: 1 vial Biotinylated mouse Urocortin 2
    4. SA-HRP Solution Liquid: 1 bottle (12 mL) HRP-labeled streptoavidin
    5. Substrate Buffer Liquid: 1 bottle (24 mL) 0.015% Hydrogen Peroxide
    6. OPD Tablet: 2 tablets o-Phenylenediamine dihydrochloride
    7. Stop Solution Liquid: 1 bottle (12 mL) 1 M H2SO4
    8. Buffer Solution Liquid: 1 bottle (15 mL) Citrate buffer
    9. Wash Solution Liquid: 1 bottle (50 mL) Concentrated saline Concentrate
    10. Plate Seal: 3 sheets
    Material not included
    Photometer for microtiter plate (plate reader), which can read absorbance up to 2.5 at 490 or 492 nm
    Rotator for microtiter plate
    Washing device for microtiter plate and dispenser with aspiration system
    Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
    Glass test tubes for preparation of Calibrator Solution
    Graduated cylinder (1,000 mL)
    Distilled water or de-ionized water
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  • Plate
    Pre-coated
    Reagent Preparation
    1. Preparation of Calibrator Solutions: Reconstitute the Urocortin 2 Calibrator (lyophilized mouse Urocortin 2, 200 ng /vial) with 1 mL of Buffer Solution, giving a 200 ng/mL Calibrator Solution after reconstitution. 0.1 mL of the recon- stituted Calibrator Solution is diluted with 0.2 mL of Buffer Solution to yield a 66.7 ng/mL Calibrator Solution. Repeat the serial dilution to make Calibrator Solutions at 22.2, 7.41, 2.47, and 0.82 ng/mL. Buffer Solution is used as the zero calibrator (0 ng/mL). Note: Calibrator Solution must be prepared immediately before assay. Use clean test tubes or vessels.

      2. Preparation of Labeled Antigen: Reconstitute Labeled Antigen with 6 mL of distilled water. Note: Labeled Antigen must be prepared immediately before assay. Use clean test tubes or vessels.

      3. Preparation of Substrate Solution: Dissolve OPD Tablet in 11 mL of Substrate Buffer. Note: Substrate Solution must be prepared immediately before use. Use clean test tubes or vessels.

      4. Preparation of Wash Solution: Dilute 50 mL of Wash Solution Concentrate to 1,000 mL with distilled or de-ionized water. Note: Diluted Wash Solution is stable for 6 months when stored at 4°C. During storage of the Wash Solution Concentrate at 4°C, precipitates may be observed, however, they will dissolve when diluted.

      5. Other reagents are ready for use.
    Assay Procedure
    1. Warm the reagents and samples to room temperature (20
      30°C) before beginning the test.
      2. Add 0.35 mL of diluted Wash Solution into the wells and aspirate the Washing Solution in the wells. Repeat this washing procedure twice, for a total of 3 wash steps. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
      3. Add 25 µL Buffer Solution into the wells, then add 20 µL of the prepared Calibrator Solutions (0, 0.82, 2.47, 7.41, 22.22, 66.7, 200 ng/mL) or samples into wells. Add 50 µL of Labeled Antigen into the wells. The total pipetting time of calibrator solutions and samples for a whole plate should not exceed 30 minutes.
      4. Cover the plate with the Plate Seal and incubate at 4°C for 16-18 hours. Plate rotator not needed for this step.
      5. Incubate plate for 40 minutes at room temperature. Plate rotator not needed for this step.
      6. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells four times with approximately 0.35 mL/well of diluted Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
      7. Pipette 100 µL of SA-HRP Solution into each of the wells.
      8. Cover the plate with a Plate Seal and incubate at room temperature for 2 hours. During the incubation, the plate should be rotated on a plate rotator (~ 100 rpm).
      9. Remove the Plate Seal, aspirate and wash the wells four times with approximately 0.35 mL/well of diluted Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
      10. Add 100 µL of Substrate Solution into the wells, cover the plate with a Plate Seal and incubate for 20 minutes at room temperature. Plate rotator not needed for this step.
      11. Add 100 µL of Stop Solution into the wells to stop the reaction.
      12. Read the optical absorbance of the wells at 490 or 492 nm. The optical absorbance of reaction solution in wells should be read as soon as possible after stopping the color reaction. Note: Test all samples in duplicate.
    Calculation of Results

    The dose-response curve of this assay fits best to a 4 or 5-parameter logistic equation. The results of unkonwn samples can be calculated with any computer program having a 4 or 5-parameter logistic function. Otherwise calculate mean absorbance values of wells containing calibrators and plot a calibration curve on semi logarithmic graph paper (abscissa: concentation of calibrator, ordinate, absorbance values). Use the average absorbance of each sample to determine the corresponding value by simple interpolation from this calibration curve. When a sample value exceeds 200 ng/mL, it must be diluted with Buffer Solution and re-assayed until the sample value is within the assay range.

    Restrictions
    For Research Use only
  • Storage
    4 °C
    Storage Comment
    Store kit at 4°C. This kit can be used dividedly in strips of the plate. In such case, the rest of reconstituted reagents (calibrator and labeled antigen) should be stored at or below -30°C (stable for 1 month).
    Expiry Date
    1 month
  • Target See all Urocortin 2 (UCN2) ELISA Kits
    Urocortin 2 (UCN2)
    Alternative Name
    Urocortin 2 (UCN2 Products)
    Synonyms
    Ucn3 ELISA Kit, SRP ELISA Kit, UCN-II ELISA Kit, UCNI ELISA Kit, UR ELISA Kit, URP ELISA Kit, Ucn II ELISA Kit, Ucn-2 ELISA Kit, ucn-II ELISA Kit, urocortin 2 ELISA Kit, Ucn2 ELISA Kit, UCN2 ELISA Kit, ucn2 ELISA Kit
    Pathways
    Positive Regulation of Peptide Hormone Secretion, Negative Regulation of Hormone Secretion, cAMP Metabolic Process
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