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TNF alpha ELISA Kit

TNF alpha Reactivity: Rat Colorimetric Sandwich ELISA Cell Lysate, Tissue Lysate
Catalog No. ABIN625217
  • Target See all TNF alpha ELISA Kits
    TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))
    Reactivity
    • 14
    • 12
    • 9
    • 5
    • 4
    • 4
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Rat
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Application
    ELISA
    Purpose
    Rat TNF alpha ELISA Kit for cell and tissue lysate samples.
    Sample Type
    Cell Lysate, Tissue Lysate
    Analytical Method
    Quantitative
    Specificity
    The antibody pair provided in this kit recognizes rat TNF-alpha.
    Cross-Reactivity (Details)
    This ELISA kit shows no cross-reactivity with any of the cytokines tested (e.g., rat CINC-2, CINC-3, CNTF, Fractalkine, IL-1alpha, IL- 1beta, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, Leptin, Lix, MCP-1, MIP-3alpha, beta- NGF, TIMP-1, VEGF).
    Sensitivity
    25 pg/mL
    Characteristics
    • 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
    Components
    • 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
    Material not included
    • 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
    • Cell lysate buffer
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  • Sample Volume
    100 μL
    Plate
    Pre-coated
    Protocol
    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.
    Reagent Preparation
    1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
      2. Sample dilution: Tissue lysate and cell lysate sample should be diluted at least 5-fold with 1x Sample Diluent Buffer.
      3. Sample Diluent Buffer (Item D) and Assay Diluent (Item E) should be diluted 5-fold with deionized or distilled water before use.
      4. Preparation of standard: Briefly spin the vial of Item C. Add 400 µL 1x Sample Diluent Buffer (Item D, should be diluted 5-fold with deionized or distilled water before use) into Item C vial to prepare a 100 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 100 µL TNF-alpha standard from the vial of Item C, into a tube with 400 µL Sample Diluent Buffer to prepare a 20,000 pg/mL stock standard solution. Pipette 400 µL 1x Sample Diluent Buffer into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. 1x Sample Diluent Buffer serves as the zero standard (0 pg/mL). 200 µL 100 µL standard + 400 µL 200myl 200 µL 200 µL 200 µL 20,000 6,667 2,222 740.7 246.9 82.30 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 Diuent 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 and used in step 4 of Part VI Assay Procedure.
      7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) before use. HRP-Streptavidin concentrate should be diluted 200-fold with 1x Assay Diluent. 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 to prepare a 200-fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.
      8. Cell lysate buffer should be diluted 2-fold with deionized or distilled water (for cell lysate and tissue lysate).
    Assay Procedure
    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.
    Calculation of Results

    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. Sample Diluent Buffer Rat TNF-alpha concentration (pg/mL) O D =4 50 n m 0.1 1 10 10 100 1,000 10,000 100,000
    Sensitivity: The minimum detectable dose of TNF-alpha is typically less than 25 pg/mL.
    Recovery: Recovery was determined by spiking various levels of Rat TNF-alpha into Rat tissue lysate and cell lysate. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Tissue lysate 92.48 80-104 Cell lysate 93.17 81-105
    Linearity: Sample Type Tissue Cell Lysate lysate 1:2 Average % of 90 88 Expected Range ( %) 80-103 76-102 1:4 Average % of 94 92 Expected Range ( %) 84-106 83-104
    Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

    Assay Precision
    Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %
    Restrictions
    For Research Use only
  • Handling Advice
    Avoid repeated freeze-thaw cycles.
    Storage
    -20 °C
    Storage Comment
    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.
    Expiry Date
    6 months
  • Iwatsuki, Arai, Ota, Kato, Natsume, Kurimoto, Yamamoto, Hirata: "Targeting anti-inflammatory treatment can ameliorate injury-induced neuropathic pain." in: PLoS ONE, Vol. 8, Issue 2, pp. e57721, (2013) (PubMed).

    Bijjem, Padi, lal Sharma: "Pharmacological activation of heme oxygenase (HO)-1/carbon monoxide pathway prevents the development of peripheral neuropathic pain in Wistar rats." in: Naunyn-Schmiedeberg's archives of pharmacology, Vol. 386, Issue 1, pp. 79-90, (2013) (PubMed).

    Brouckaert, Libert, Everaerdt, Takahashi, Cauwels, Fiers: "Tumor necrosis factor, its receptors and the connection with interleukin 1 and interleukin 6." in: Immunobiology, Vol. 187, Issue 3-5, pp. 317-29, (1993) (PubMed).

    Bonavida: "Immunomodulatory effect of tumor necrosis factor." in: Biotherapy (Dordrecht, Netherlands), Vol. 3, Issue 2, pp. 127-33, (1991) (PubMed).

    Blankenstein, Qin, Uberla, Müller, Rosen, Volk, Diamantstein: "Tumor suppression after tumor cell-targeted tumor necrosis factor alpha gene transfer." in: The Journal of experimental medicine, Vol. 173, Issue 5, pp. 1047-52, (1991) (PubMed).

  • Target See all TNF alpha ELISA Kits
    TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))
    Alternative Name
    TNF-alpha (TNF alpha Products)
    Synonyms
    DIF ELISA Kit, TNF-alpha ELISA Kit, TNFA ELISA Kit, TNFSF2 ELISA Kit, RATTNF ELISA Kit, Tnfa ELISA Kit, tnf ELISA Kit, TNF-a ELISA Kit, TNFalpha ELISA Kit, Tnfsf1a ELISA Kit, TNFa ELISA Kit, cTNF ELISA Kit, Tnf-alpha ELISA Kit, tnfa-like ELISA Kit, TNF-ALPHA ELISA Kit, dif ELISA Kit, tnfa ELISA Kit, xtnf ELISA Kit, tnfsf2 ELISA Kit, tnf-alpha ELISA Kit, Cachectin ELISA Kit, tumor necrosis factor ELISA Kit, tumor necrosis factor b (TNF superfamily, member 2) ELISA Kit, tumor necrosis factor alpha ELISA Kit, tumor necrosis factor a (TNF superfamily, member 2) ELISA Kit, TNF ELISA Kit, Tnf ELISA Kit, tnf ELISA Kit, tnfb ELISA Kit, tnf-alpha ELISA Kit, LOC103694380 ELISA Kit, tnfa ELISA Kit
    Background
    Tumor necrosis factor (Cachectin) (TNF-alpha) (Tumor necrosis factor ligand superfamily member 2) (TNF-a)
    Gene ID
    103694380, 24835
    UniProt
    P16599
    Pathways
    NF-kappaB Signaling, Apoptosis, Caspase Cascade in Apoptosis, TLR Signaling, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Inflammasome
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