TGFB1 Kit ELISA

N° du produit ABIN612788

Détail du produit TGFB1 Kit ELISA

Antigène: TGFB1 (Transforming Growth Factor, beta 1 (TGFB1))

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Seuil minimal de détection: 30 pg/mL

Application: ELISA

Fonction: The AssayMax Human TGF-1 ELISA kit is designed for detection of TGF-1 in cell culture supernatants

Marque: AssayMax

Type d'échantillon: Cell Culture Supernatant

Analytical Method: Quantitative

Specificité: This assay recognizes both natural and recombinant human TGF-1.

Ingrédients: TGF-1 Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a murine monoclonal antibody against TGF-1. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. TGF-1 Standard: Human TGF-1 in a buffered protein base (2 ng, lyophilized). 1 Biotinylated TGF-1 Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against TGF-1 (80µl). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Matériel non inclus: Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water

Information d'application

Volume d'échantillon: 50 μL

Durée du test: 4.5 h

Plaque: Pre-coated

Protocole: This assay employs a quantitative sandwich enzyme immunoassay technique that measures TGF-1 in 4.5 hours. A murine monoclonal antibody specific for human TGF-1 has been pre-coated onto a microplate. TGF-1 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human TGF-1, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Préparation des réactifs:

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 2 ng of human TGF-1 Standard with 1 ml of EIA Diluent to generate a solution of 2 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the TGF-1 standard solution twofold with equal volume of EIA Diluent to produce 1, 0.5, 0.25, 0.125, 0.0625 and 0.031 ng/ml. EIA Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [TGF-1] (ng/ml) P1 Standard (2 ng/ml) 2.000 P2 1 part P1 + 1 part EIA Diluent 1.000 P3 1 part P1 + 1 part EIA Diluent 0.500 P4 1 part P1 + 1 part EIA Diluent 0.250 P5 1 part P1 + 1 part EIA Diluent 0.125 P6 1 part P1 + 1 part EIA Diluent 0.063 P7 1 part P1 + 1 part EIA Diluent 0.031 P8 EIA Diluent 0.000 Biotinylated TGF-1 Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C.

Prélèvement de l'échantillon: Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles.

Procédure de l'essai:

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated TGF-1 Antibody to each well and incubate for two hours. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Chromogen Substrate per well and incubate for approximately 15 minutes or till the optimal blue color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately.

Calcul des résultats:

Calculate the mean value of the triplicate readings for each standard and sample. To generate a Standard Curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Précision du teste: Intra-assay and inter-assay coefficients of variation were 5.2% and 7.0% respectively.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: The kit should not be used beyond the expiration date.

Stock: 4 °C/-20 °C

Stockage commentaire: Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened EIA Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along with zip-seal. May be stored for up to 1 month in a vacuum desiccator.

Détail du antigène

Antigène: TGFB1 (Transforming Growth Factor, beta 1 (TGFB1))

Autre désignation: Transforming Growth Factor-beta 1 (TGF-1) (TGFB1 Produits)

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Sujet: Transforming growth factor-beta 1 (TGF-1) is one of the transforming growth factor beta (TGF- ) family cytokines and exerts pleiotropic effects upon a wide variety of cell types. TGF-1 has been demonstrated to be of fundamental importance in the development, physiology and pathology of the vascular system. It is known to maintain a balance between apoptosis and cellular dysfunction. Over-expression of TGF-1 is the cellular change associated with abnormal extracellular matrix deposition in nodular glomerulosclerosis , and may be a pathogenetic mechanism in tumor progression. High serum levels of TGF-1 probably mirror an anti-inflammatory response, which might play a role in controlling the systemic immune response.

Pathways: EGFR Signaling Pathway, Dopaminergic Neurogenesis, Cellular Response to Molecule of Bacterial Origin, Glycosaminoglycan Metabolic Process, Regulation of Leukocyte Mediated Immunity, Regulation of Muscle Cell Differentiation, Positive Regulation of Immune Effector Process, Cell-Cell Junction Organization, Production of Molecular Mediator of Immune Response, Ribonucleoside Biosynthetic Process, Skeletal Muscle Fiber Development, Regulation of Carbohydrate Metabolic Process, Protein targeting to Nucleus, Autophagy, Cancer Immune Checkpoints