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CNTF ELISA Kit

CNTF Reaktivität: Ratte Colorimetric Sandwich ELISA 10-2000 pg/mL Cell Culture Supernatant, Plasma, Serum
Produktnummer ABIN1979805
  • Target Alle CNTF ELISA Kits anzeigen
    CNTF (Ciliary Neurotrophic Factor (CNTF))
    Reaktivität
    • 4
    • 3
    • 3
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    Ratte
    Nachweismethode
    Colorimetric
    Methodentyp
    Sandwich ELISA
    Detektionsbereich
    10-2000 pg/mL
    Untere Nachweisgrenze
    10 pg/mL
    Applikation
    ELISA
    Verwendungszweck
    Rat CNTF ELISA Kit for cell culture supernatants, plasma, and serum samples.
    Proben
    Plasma, Cell Culture Supernatant, Serum
    Analytische Methode
    Quantitative
    Spezifität
    This ELISA kit shows no cross-reactivity with any of the cytokines tested: rat CINC-2, CINC-3, Fractalkine, IL-1 alpha, IL-1 beta, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, Leptin, Lix, MCP-1, MIP-3 alpha, beta-NGF, TIMP- 1, TNF-alpha, VEGF.
    Sensitivität
    < 10 pg/mL
    Produktmerkmale
    • 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
    Bestandteile
    • Pre-Coated 96-well Strip Microplate
    • Wash Buffer
    • Stop Solution
    • Assay Diluent(s)
    • Lyophilized Standard
    • Biotinylated Detection Antibody
    • Streptavidin-Conjugated HRP
    • TMB One-Step Substrate
    Benötigtes Material
    • Distilled or deionized water
    • Precision pipettes to deliver 2 μL to 1 μL volumes
    • Adjustable 1-25 μL pipettes for reagent preparation
    • 100 μL and 1 liter graduated cylinders
    • Tubes to prepare standard and sample dilutions
    • Absorbent paper
    • Microplate reader capable of measuring absorbance at 450nm
    • Log-log graph paper or computer and software for ELISA data analysis
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  • Applikationshinweise
    Recommended Dilution for serum and plasma samples2 fold
    Probenmenge
    100 μL
    Plattentyp
    Pre-coated
    Protokoll
    1. Prepare all reagents, samples and standards as instructed in the manual.
    2. Add 100 μL of standard or sample to each well.
    3. Incubate 2.5 h at RT or O/N at 4 °C.
    4. Add 100 μL of prepared biotin antibody to each well.
    5. Incubate 1 h at RT.
    6. Add 100 μL of prepared Streptavidin solution to each well.
    7. Incubate 45 min at RT.
    8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
    9. Incubate 30 min at RT.
    10. Add 50 μL of Stop Solution to each well.
    11. Read at 450 nm immediately.
    Aufbereitung der Reagenzien
    1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
      2. Sample dilution: If your samples need to be diluted, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. 1x Assay Diluent B (Item E) should be used for dilution of culture supernatants. Suggested dilution for normal serum/plasma: 2 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
      3. Assay Diluent B should be diluted 5-fold with deionized or distilled water.
      4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 40 µL CNTF standard from the vial of Item C, into a tube with 960 µL Assay Diluent A or 1x Assay Diluent B to prepare a 2,000 pg/mL stock standard solution. Pipette 400 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 200 µL 200myl 200 µL 200 µL 200 µL 40 µL standard + 960 µL 2000 666.7 222.2 74.07 24.69 8.23 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
      5. If the Wash Concentrate (20x) (Item B) 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.
      6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
      7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP- Streptavidin concentrate should be diluted 200-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 50 µL of HRP-Streptavidin concentrate into a tube with 10 ml 1x Assay Diluent B to prepare a 200-fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.
    Testdurchführung
    1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
      2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
      3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good 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 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
      5. Discard the solution. Repeat the wash as in step
      6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
      7. Discard the solution. Repeat the wash as in step
      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.
      9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.
    Ergebnisberechnung

    Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
    Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent A Rat CNTF concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 (n m ) 0.01 0.1 1 10 Assay Diluent B Rat CNTF concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 (n m ) 0.01 0.1 1 10
    Sensitivity: The minimum detectable dose of CNTF is typically less than 10 pg/mL.
    Recovery: Recovery was determined by spiking various levels of Rat CNTF into Rat serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 105.6 93-112 Plasma 104.2 92-110 Cell culture media 97.3 89-108
    Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 95 97 94 Range ( %) 85-104 86-105 82-102 1:4 Average % of Expected 97 101 96 Range ( %) 86-106 90-106 85-104
    Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

    Testpräzision
    Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %
    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Handhabung
    Avoid repeated freeze-thaw cycles.
    Lagerung
    -20 °C
    Informationen zur Lagerung
    The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.
    Haltbarkeit
    6 months
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  • Target Alle CNTF ELISA Kits anzeigen
    CNTF (Ciliary Neurotrophic Factor (CNTF))
    Andere Bezeichnung
    CNTF (CNTF Produkte)
    Synonyme
    GPA ELISA Kit, CNTF ELISA Kit, HCNTF ELISA Kit, AI429687 ELISA Kit, ciliary neurotrophic factor ELISA Kit, CNTF ELISA Kit, Cntf ELISA Kit
    Hintergrund
    Ciliary neurotrophic factor (CNTF)
    Gen-ID
    25707
    UniProt
    P20294
    Pathways
    JAK-STAT Signalweg
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