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TGFB2 ELISA Kit (Transforming Growth Factor, beta 2) ELISA Kit

TGFB2 Reactivity: Human Colorimetric Sandwich ELISA 15-6000 pg/mL Plasma
Pubmed (9)
Catalog No. ABIN625095
$480.70
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  • Target
    Reactivity
    Human
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Detection Range
    15-6000 pg/mL
    Minimum Detection Limit
    15 pg/mL
    Application
    ELISA
    Purpose
    Human TGF beta 2 ELISA Kit for cell culture supernatants, plasma, and serum samples.
    Sample Type
    Plasma, Cell Culture Supernatant, Serum
    Analytical Method
    Quantitative
    Specificity
    This ELISA kit shows no cross-reactivity with any of the cytokines tested: Human BDNF, BLC, ENA-78, FGF-4, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, I-309, IP-10, G-CSF, GM-CSF, IFN-gamma, Leptin (OB), MCP-1, MCP- 2, MCP-3, MDC, MIP-1 alpha, MIP-1 beta, MIP-1 delta, PARC, PDGF, RANTES, SCF, TARC, TGF-beta1, TGF-beta3, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.
    Sensitivity
    < 15 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
  • Application Notes
    Recommended Dilution for serum and plasma samples2 fold after treatment (see activation steps on page 6)
    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: 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 and urine.
      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. Add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium and urine) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 80 µL TGF-beta2 standard from the vial of Item C, into a tube with 586.7 µL Assay Diluent A or 1x Assay Diluent B to prepare a 6,000 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 200 µL 200 µL 200 µL 200 µL 80 µL standard + 586.7 µL 200myl 6000 2000 666.6 222.2 74.07 24.69 8.23 0 pg/mL 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) before use. HRP-Streptavidin concentrate should be diluted 500-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 20 µL of HRP-Streptavidin concentrate into a tube with 10 ml 1x Assay Diluent B to prepare a 500-fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). Mix well. ACTIVATION REAGENT PREPARATION To activate latent TGF-beta2 to the immunoreactive form, prepare the following solutions for activation and neutralization. The solutions may be stored in polypropylene bottles at room temperature for up to one month. Use polypropylene test tubes. Notes: Do not activate the kit standards. The kit standards contain active rhTGF-beta2. 1 N HCl (100 ml) - Slowly add 8.33 mL of 12 N HCl into 91.67 ml deionized water. Mix bottle. 1.2 N NaOH/0.5 M HEPES (100 ml) - Slowly add 12 ml of 10 N NaOH into 75 mL deionized water. Mix bottle. Add 11.9 g HEPES. Mix through. Bring final volume to 100 mL with deionized water. ACTIVATION PROCEDURE To activate latent TGF-beta2 to the immunoreactive form, follow the activation procedure. Use polypropylene test tubes. Notes: Do not activate the kit standards. The kit standards contain active rhTGF-beta2. 1. Add 25 µL 1 N HCl into 125 µL sample. Mix through.
      2. Incubate 10 minutes at room temperature.
      3. Add 25 µL 1.2 N NaOH/0.5 M HEPES. Mix through.
      4. Add 800 µL Assay Diluent A (for serum/plasma) or 1x Assay Diluent B (for cell culture supernatants/urine). Mix through and assay within 2 hours. Note: The concentration read off the standard curve must be multiplied by the dilution factor, 7.8. If samples generate values higher than the highest standard, further dilute the samples after activation with the 1x Assay diluent B.
    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. Assay Diluent A Human TGF-beta-2 concentration (pg/mL) O D =4 50 n m 0.01 0.1 1 10 0 10 100 1,000 10,000 Assay Diluent B Human TGF-beta-2 concentration (pg/mL) O D =4 50 n m 0.01 0.1 1 10 0 10 100 1,000 10,000
    Sensitivity: The minimum detectable dose of TGF- beta2 is typically less than 15 pg/mL.
    Recovery: Recovery was determined by spiking various levels of human TGF-beta2 into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 94.74 82-104 Plasma 95.53 83-103 Cell culture media 96.14 84-105
    Linearity: Sample Type Serum Plasma Cell culture media 1:2 Average % of Expected 93 94 92 Range ( %) 84-103 83-103 84-103 1:4 Average % of Expected 96 97 95 Range ( %) 85-104 84-102 85-103
    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
  • Xing, Xiao, Lu, Zhu, He, Huang, Lopez, Wong, Ju, Tian, Zhang, Xu, Wang, Li, Karin, Ren: "GFI1 downregulation promotes inflammation-linked metastasis of colorectal cancer." in: Cell death and differentiation, Vol. 24, Issue 5, pp. 929-943, 2018 (PubMed).

    Kimura, Hayashi, Mizuno, Oike: "Endothelium-dependent epithelial-mesenchymal transition of tumor cells: exclusive roles of transforming growth factor β1 and β2." in: Biochimica et biophysica acta, Vol. 1830, Issue 10, pp. 4470-81, 2013

    Kimura, Hayashi, Mizuno, Oike: "Endothelium-dependent epithelial-mesenchymal transition of tumor cells: exclusive roles of transforming growth factor β1 and β2." in: Biochimica et biophysica acta, Vol. 1830, Issue 10, pp. 4470-81, 2013

    Kimura, Hayashi, Mizuno, Oike: "Endothelium-dependent epithelial-mesenchymal transition of tumor cells: exclusive roles of transforming growth factor β1 and β2." in: Biochimica et biophysica acta, Vol. 1830, Issue 10, pp. 4470-81, 2013

    Kimura, Hayashi, Mizuno, Oike: "Endothelium-dependent epithelial-mesenchymal transition of tumor cells: exclusive roles of transforming growth factor ?1 and ?2." in: Biochimica et biophysica acta, Vol. 1830, Issue 10, pp. 4470-81, 2013 (PubMed).

    De Barros, Dehez, Arnaud, Barreau, Cazavet, Perez, Galinier, Casteilla, Planat-Bénard: "Aging-related decrease of human ASC angiogenic potential is reversed by hypoxia preconditioning through ROS production." in: Molecular therapy : the journal of the American Society of Gene Therapy, Vol. 21, Issue 2, pp. 399-408, 2013 (PubMed).

    Xue, Restuccia, Lan, Hynx, Dirnhofer, Hess, Rüegg, Hemmings: "Akt/PKB-mediated phosphorylation of Twist1 promotes tumor metastasis via mediating cross-talk between PI3K/Akt and TGF-? signaling axes." in: Cancer discovery, Vol. 2, Issue 3, pp. 248-59, 2012 (PubMed).

    Steed, Trumpower, Duffy, Smith, Marshall, Rupp, Robson: "Amnion-derived cellular cytokine solution: a physiological combination of cytokines for wound healing." in: Eplasty, Vol. 8, pp. e18, 2008 (PubMed).

    Barnard, Lyons, Moses: "The cell biology of transforming growth factor beta." in: Biochimica et biophysica acta, Vol. 1032, Issue 1, pp. 79-87, 1990 (PubMed).

  • Target
    Alternative Name
    TGF-beta 2 (TGFB2 ELISA Kit Abstract)
    Synonyms
    LDS4, TGF-beta2, tgf-beta2, tgfb2-A, BB105277, Tgf-beta2, Tgfb-2, TGFB2, TGFbeta2, MGF, TGF-B2, transforming growth factor beta 2, transforming growth factor beta 2 L homeolog, transforming growth factor, beta 2, TGFB2, tgfb2.L, Tgfb2, tgfb2
    Background
    TGF-beta exists in at least five isoforms, known as TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5. Their amino acid sequences display homologies on the order of 70-80%. The various TGF-beta isotypes share many biological activities and their actions on cells are qualitatively similar in most cases although there are a few examples of distinct activities. TGF-beta2 is the only variant that does not inhibit the growth of endothelial cells. TGF-beta2 and TGF-beta3 inhibit the survival of cultured embryonic chick ciliary ganglionic neurons. The Human TGF-beta2 ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human TGF-beta2 in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human TGF-beta2 coated on a 96-well plate. Standards and samples are pipetted into the wells and TGF-beta2 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human TGF-beta2 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of TGF-beta2 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.
    Gene ID
    7042
    UniProt
    P61812
    Pathways
    Cell-Cell Junction Organization, Production of Molecular Mediator of Immune Response, Protein targeting to Nucleus
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