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QIA61 TGF-α ELISA Kit

TGF-α ELISA Kit: SDB (Sicherheitsdatenblätter), Analysenzertifikate und Qualitätszertifikate, Dossiers, Broschüren und andere verfügbare Dokumente.
Detection Methods: Colorimetric
QIA61
  
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      Übersicht

      Replacement Information

      Key Spec Table

      Species ReactivityDetection Methods
      HColorimetric
      Description
      Overview

      This product has been discontinued.
      Catalogue NumberQIA61
      Brand Family Calbiochem®
      Application Data
      The mean signal of each standard run in replicates of three in eight assays using two different lots of plates and two different lots of detector antibody.
      Materials Required but Not Delivered• 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips.
      • Wash bottle or multichannel dispenser for washing.
      • Microcentrifuge tubes or other appropriate containers for mixing low volumes.
      • 1 liter graduated cylinder.
      • Deionized H2O.
      • Plastic wrap.
      • Adhesive tape.
      • Spectrophotometer capable of measuring absorbance in 96 well plates at a wavelength of 490 nm. Use of a 630 nm reference beam is suggested.
      • Disposable paper towels.
      • 2 µm syringe filter and syringe.
      References
      ReferencesEllis, D.L., et al. 1990. J. Invest. Dermatol. 95, 27.
      Katoh, M., et al. 1990. Biochem. Biophys. Res. Commun. 167, 1065.
      Derynck, R., 1988. Cell 54, 593.
      Coffey, R.J., et al. 1987. Nature 328, 817.
      Yeh, Y.C., et al. 1987. Cancer Res. 47, 896.
      Derynck, R., 1986. J. Cell Biochem. 32, 203.
      Samsoondar, J., et al. 1986. J. Biol. Chem. 261, 14408.
      Lee, D.C., et al. 1985. Molec. Cell. Biol. 5, 3644.
      Todaro, G.J., et al. 1985. In Genes and Protein in Oncogenesis, Vogel, H., Weinstein, I.B. (Eds.) New York, Raven Press pp. 165.
      Anzano, M.A., et al. 1983. Proc. Natl. Acad. Sci. USA 80, 6264.
      Marquardt, H., et al. 1983. Proc. Natl. Acad. Sci. USA 80, 4684.
      Reynolds, F.H., Jr., et al. 1983. Nature 292, 259.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsAntibody Coated 96-Well Plate, TGF-α Standards, Reporter Antibody, Peroxidase Conjugate, Peroxidase Substrate, Stop Solution, Rinse Buffer, Antigen Extraction Agent, Assay Buffer, Plate Sealers, and a user protocol.
      Quality LevelMQ100
      Applications
      Biological Information
      Assay range0-1000 pg/ml
      Assay time4 h
      Sample TypeSerum, cell lysates, tissue culture supernatants, and plasma samples
      Species Reactivity
      • Human
      Physicochemical Information
      Sensitivity5 pg/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® TGFα ELISA is a non isotopic immunoassay for the in vitro quantitation of human Transforming Growth Factor Alpha (TGFα) in serum, tissue culture media, and other fluids.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage -20°C
      Storage ConditionsUpon receipt, standards must be stored at -20°C. All other components may be stored at -20°C or 4°C (do not refreeze after thawing). Do not expose reagents to excessive light. Allow kit reagents to warm to room temperature before use. Reagent Stability: All of the reagents included with the TGFα ELISA have been tested for stability. Assay Buffer should not be used if cloudiness or solid matter is present. Coated plates should be stored at 4°C in the original foil bag containing a desiccant pack.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAntibody Coated 96-Well Plate, TGF-α Standards, Reporter Antibody, Peroxidase Conjugate, Peroxidase Substrate, Stop Solution, Rinse Buffer, Antigen Extraction Agent, Assay Buffer, Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Bestellnummer GTIN
      QIA61 0

      Documentation

      TGF-α ELISA Kit Analysenzertifikate

      TitelChargennummer
      QIA61

      Literatur

      Übersicht
      Ellis, D.L., et al. 1990. J. Invest. Dermatol. 95, 27.
      Katoh, M., et al. 1990. Biochem. Biophys. Res. Commun. 167, 1065.
      Derynck, R., 1988. Cell 54, 593.
      Coffey, R.J., et al. 1987. Nature 328, 817.
      Yeh, Y.C., et al. 1987. Cancer Res. 47, 896.
      Derynck, R., 1986. J. Cell Biochem. 32, 203.
      Samsoondar, J., et al. 1986. J. Biol. Chem. 261, 14408.
      Lee, D.C., et al. 1985. Molec. Cell. Biol. 5, 3644.
      Todaro, G.J., et al. 1985. In Genes and Protein in Oncogenesis, Vogel, H., Weinstein, I.B. (Eds.) New York, Raven Press pp. 165.
      Anzano, M.A., et al. 1983. Proc. Natl. Acad. Sci. USA 80, 6264.
      Marquardt, H., et al. 1983. Proc. Natl. Acad. Sci. USA 80, 4684.
      Reynolds, F.H., Jr., et al. 1983. Nature 292, 259.

      Technische Informationen

      Titel
      QIA61 TGFα- ELISA Kit QIA61

      Literaturstellen

      Titel
    • Mark R. Bush, et al. (1998) Evidence of Juxtacrine Signaling for Transforming Growth Factor a in Human Endometrium. 59, 1522-1529.
      		•
      Anwenderprotokoll

      Revision19-March-2013 JSW
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon receipt, standards must be stored at -20°C. All other components may be stored at -20°C or 4°C (do not refreeze after thawing). Do not expose reagents to excessive light. Allow kit reagents to warm to room temperature before use. Reagent Stability: All of the reagents included with the TGFα ELISA have been tested for stability. Assay Buffer should not be used if cloudiness or solid matter is present. Coated plates should be stored at 4°C in the original foil bag containing a desiccant pack.
      Intended useThe Calbiochem® TGFα ELISA is a non isotopic immunoassay for the in vitro quantitation of human Transforming Growth Factor Alpha (TGFα) in serum, tissue culture media, and other fluids.
      BackgroundTransforming growth factors (TGFs) are potent mitogenic polypeptides. Two classes of TGFs have been identified: TGFα and TGFβ. TGFα is synthesized as a 160 amino acid, transmembrane precursor. The precursor undergoes sequential external proteolytic cleavage, releasing TGFa species ranging in molecular weight from 6-25 kDa. Mature TGFα is an acid- and heat-stable 50 amino acid polypeptide of 5.5 kDa. It is secreted by a variety of transformed cells and tumors, and by a number of embryonic cells as well as some normal adult tissues including the brain and skin keratinocytes. Mature TGFα is approximately 30% homologous, at the amino acid level, to epidermal growth factor (EGF). TGFα binds to the EGF receptor, mediates tyrosine phosphorylation of the receptor, and promotes anchorage-independent growth of normal rat fibroblasts in soft agar in the presence of transforming growth factor-β. Transformation of cells, in vitro, by certain viruses including SV40 and polyoma virus, as well as transformation by the activated ras oncogene can lead to increased secretion of TGFα. TGFα may play a role in tumor formation by an autocrine/paracrine mechanism whereby TGFα promotes and sustains the transformed character of the cells from which it is secreted as well as that of neighboring cells. The biological relevance of TGFα in tumor development is supported by reports that multiple molecular weight forms of TGFα may be found in the urine of cancer patients in amounts which differ from those found in normal urine samples. TGFα may also play a role in normal development including wound healing. The major difficulty impeding the investigation of the normal and pathological roles of TGFα in vivo has been the use of activity-based biological assays that do not distinguish between TGFα and other related molecules (e.g., EGF). The Calbiochem® TGFa ELISA is highly specific and can detect less than 10 pg/ml of human TGFα in a variety of biological fluids and tissue culture media.
      Principles of the assayThe Calbiochem® TGFα ELISA is a "sandwich" enzyme immunoassay which utilizes affinity purified goat polyclonal antibodies specific for mammalian TGFα. Polyclonal TGFα antibodies have been immobilized onto the surface of the plastic wells provided in the kit. The sample to be assayed and biotinylated goat TGFα antibodies, are added to the wells, and allowed to incubate for a period of time. During this time, any TGFα present in the sample binds to both the capture antibody on the plate, and to the reporter antibody (biotinylated) in solution. Unbound material is washed away. The reporter antibody is bound, in turn, by streptavidin-horseradish peroxidase.

      The horseradish peroxidase catalyzes the conversion of the chromogenic substrate o-phenylenediamine dihydrochloride from a colorless solution to a light amber solution, the intensity of which is proportional to the amount of TGFα bound to the plate. After stopping further color development, the colored reaction product is measured using a spectrophotometer. Quantitation is achieved by the construction of a standard curve using known concentrations of TGFα. By comparing the absorbance obtained from a sample containing an unknown amount of TGFα with that obtained from the standards, the concentration of TGFα in the sample can be determined.
      Materials providedStandards should be assayed in duplicate. A standard curve must be performed on the same plate and at the same time as the samples. The TGFα ELISA provides sufficient reagents to run two sets of standard curves, and 41 samples (if assayed in duplicate all at once using one standard curve), or 34 samples (if assayed on two separate occasions using two standard curves).

      • Anti-TGFα Coated Plate (Kit Component No. JA1857-1EA): 96 removable wells coated with TGFα polyclonal antibody.
      • TGFα Standards (Kit Component No. JA1563): two vials containing lyophilized, synthetic, biologically active TGFα.
      • Reconstituted standards should be discarded after one use.
      • Plate Sealers (Kit Component No. ): Plate sealers are provided to cover the plate during incubations.
      • Reporter Antibody (Kit Component No. JA1856): (500 µl) 20X concentrated solution of biotinylated goat TGFα antibody.
      • Peroxidase Conjugate (Kit Component No. JA1087): (200 µl) 100X concentrated solution of streptavidin conjugated to horseradish peroxidase.
      • Peroxidase Substrate (Kit Component No. JA1086): (5 tablets) O-Phenylenediamine (OPD) and (20 ml) Substrate Diluent.
      • Stop Solution (Kit Component No. JA1616): (20 ml) 2.5 sulfuric acid.
      • Rinse Buffer (Kit Component No. JA1084): (30 ml) 10X concentrated buffer containing bovine serum albumin, a surfactant and 0.1% 2 chloroacetamide.
      • Antigen Extraction Agent (AEA) (Kit Component No. JA1855): Use as directed in sample preparation section for the extraction of TGFα from cell preparations
      • Assay Buffer (Kit Component No. JA1085): (30 ml) Buffer containing bovine serum albumin, a surfactant, protein stabilizers and 0.1% 2 chloroacetamide.
      NOTE: The amount of Assay Buffer provided is sufficient when used as directed. Use only as much Assay Buffer as needed to ensure delivery of 100 µl of sample to each well. If a large excess of diluent sample is prepared (e.g., 1 ml rather than 0.22 ml needed for each sample, in duplicate) Assay Buffer may become limiting.

      • Wash Buffer (Kit Component No. JA1617): (100 ml) 20X concentrated buffer and surfactant.
      Materials Required but not provided• 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips.
      • Wash bottle or multichannel dispenser for washing.
      • Microcentrifuge tubes or other appropriate containers for mixing low volumes.
      • 1 liter graduated cylinder.
      • Deionized H2O.
      • Plastic wrap.
      • Adhesive tape.
      • Spectrophotometer capable of measuring absorbance in 96 well plates at a wavelength of 490 nm. Use of a 630 nm reference beam is suggested.
      • Disposable paper towels.
      • 2 µm syringe filter and syringe.
      Precautions and recommendations• Store standards at -20°C. All other components may be stored at -20°C or 4°C (do not refreeze after thawing). Do not expose reagents to excessive light. Warm kit reagents to room temperature before use (let sit at room temperature ~30 min before use.)
      • Use only the wells provided with the kit.
      • Do not mix reagents from different kits.
      • The buffers and reagents used in this kit contain 2 chloroacetamide. Care should be taken to prevent direct contact with these products.
      • Do not mouth pipette or ingest any of the reagents.
      • Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled.
      • Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
      • Do not handle the substrate tablets with fingers or permit contact with skin, metal or oxidizing agents. Dispose of OPD (o-phenylenediamine) containing solutions in compliance with local regulations.
      • Wear disposable gloves and eye protection when handling Stop Solution (2.5N sulfuric acid).
      PreparationCell Lysate Protocol. Numerous extraction protocols can be used. The following protocol has been shown to work with a number of cell lines. It is provided as an example of a suitable extraction procedure, but should not be construed as necessarily being the method of choice. Users may wish to experiment with extraction procedures that work best in their hands. 1. For suspension cells, pellet by centrifugation, remove supernatant, resuspend with PBS and pellet by centrifugation. For attached cells, remove supernatant from cells (you may save the supernatant for testing in the ELISA). Wash cells once with PBS, harvest cells by scraping and gentle centrifugation. 2. Aspirate PBS leaving an intact cell pellet (at this point the cell pellet can be frozen at -80°C and lysed at a later date). We recommend for every 5 x 106 cells, resuspend the pellet in 1 ml of Resuspension Buffer (50 mM Tris containing 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, and 0.5 µg/ml of leupeptin adjusted to pH 7.4. 3. Add 20 µl of Antigen Extraction Agent (AEA provided) for every 100 µl of cell suspension. 4. Incubate 30 min on ice with occasional vortexing. 5. Transfer extracts to microcentrifuge tubes and centrifuge for 5 min at 500 x g at 4°C. 6. Aliquot cleared lysate to clean microcentrifuge tubes. The sample should be aliquotted to avoid multiple freeze/thaws. These samples are now ready for analysis according to the instructions provided in the Detailed Protocol. Samples may be stored at -20°C for future testing in the TGFα ELISA. 7. Samples found to contain greater than 1000 pg/ml TGFα (i.e., outside the range of the standard curve) must be diluted with Assay Buffer (provided), so that the TGFα concentration falls within the range spanned by the standard curve, and assayed again. 8. Appropriate negative controls should be included. For example, if TGFα is being measured in cell conditioned medium, a sample of the same medium which has not been exposed to cells should also be assayed. 9. Appropriate negative controls should be included. For example, if TGFα is being measured in cell conditioned medium, a sample of the same medium which has not been exposed to cells should also be assayed.
      Detailed protocolThe TGFα ELISA is provided with removable strips of wells so the assay can be carried out on separate occasions. Since conditions may vary, a standard curve must be determined each time the assay is performed. Both standards and samples should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross contamination of reagents or samples.
      1. Remove the appropriate number of wells from the foil pouch. Return any unused wells to the foil pouch containing the desiccant pack. SEAL TIGHTLY and store at 4°C. Let all other kit components sit at room temperature until used. Best results will be obtained using reagents at room temperature.
      2. Prepare a working solution (1X) of Rinse Buffer by adding 30 ml of 10X concentrated solution to 270 ml of deionized water. Mix well.
      3. Prepare a working solution (1X) of Wash Buffer by adding 50 ml of the 20X concentrated solution to 950 ml of deionized water. Mix well.
      4. Reconstitute Lyophilized Standard (refer to vial label for lot specific reconstitution volume of dH2O and/or Assay Buffer) to yield a stock solution of 1000 pg/ml. Let the reconstituted standard sit for 15 min, with occasional swirling. Avoid excessive agitation of the standard. Serial dilutions of the standard should be made with Assay Buffer. To make serial dilutions of the standard, obtain 6 tubes and label them, 1000, 500, 250, 125, 62.5, 0 pg/ml. Add 250 µl of Assay Buffer into each tube, except the 1000 pg/ml tube (first tube) which gets "undiluted" standard. Remove 500 µl from the undiluted, reconstituted standard vial (1000 pg/ml) and add it to the first tube. Remove 250 µl from the first tube (1000 pg/ml) and add it to the second tube (500 pg/ml) and mix gently. Repeat this procedure until you reach the fifth tube (62.5 pg/ml). The last tube (0 pg/ml) should contain only 250 µl of Assay Buffer.
      5. Dilute Reporter Antibody 1:20 in Assay Buffer to provide 55 µl of 1X solution for each of the sample and standard wells. (For example: add 60 µl to 1,140 µl of Assay Buffer to make 1.2 ml of working solution, sufficient for 20-21 wells).
      6. Add 50 µl of diluted Reporter Antibody to appropriate wells, in DUPLICATE.
      7. Add 50 µl of standards/samples to appropriate wells, in duplicate, on top of reporter antibody.
      8. Cover plate with a plate sealer and incubate at room temperature for 3 h.
      9. Dilute a sufficient amount of the Peroxidase Conjugate 1:100 in Assay Buffer to provide 100 µl of 1X solution for each of the sample and standard wells. (For example: add 11 µl to 1,089 µl of Assay Buffer to make 1.1 ml of working solution, sufficient for 10 wells). Filter with a 0.2 µm syringe filter prior to use.
      10. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
      11. Wash wells one time using 200 µl of 1X Rinse Buffer per well. Do not let the Rinse Buffer incubate on the plate for more than five min. Empty the contents of the wells by inverting the plate over a sink then tapping dry on paper towels.
      12. Add 100 µl of the 1X Peroxidase Conjugate into each well. Cover with plastic wrap and incubate 30 min at room temperature.
      13. Prepare sufficient Substrate Solution to provide 100 µl for each well. Substrate Solution is prepared by dissolving one (1) OPD tablet per 4 ml of Substrate Diluent and mixing gently. Once prepared, substrate solution should be used within 30 min. AVOID EXPOSURE TO LIGHT.
      WARNING: Do not handle substrate tablets with fingers or permit contact with skin, metal or oxidizing agents. If contacted, flush with water. Solutions should be disposed of in compliance with local regulations.
      14. Wash wells 3 times with 1X Wash Buffer making sure each well is filled completely.
      15. Wash wells one time using 200 µl of 1X Rinse Buffer per well. Do not let the Rinse Buffer incubate on the plate for more than five min. Empty the contents of the wells by inverting the plate over a sink then tapping dry on paper towels.
      16. Add 100 µl of Substrate Solution to each well and incubate IN THE DARK at room temperature for 30 min.
      17. Add 100 µl of Stop Solution to each well in the same order as the previously added Substrate Solution.
      18. Measure absorbance in each well using a spectrophotometric plate reader at a wavelength of 490 nm. Wells must be read within 30 min of adding the Stop Solution.
      Calculations1. Average the duplicate absorbance values for each standard, including the zero, and all sample values. 2. On graph paper, plot the mean absorbance values for each of the standards on the Y axis, versus the concentration of each standard (pg/ml) on the X axis. 3. Determine the concentration of unknowns by interpolation from the standard curve. There are a variety of plate reader software packages available (Softmax, Molecular Devices Corporation, Menlo Park, CA; KinetiCalc, BioTek Instruments, Inc. Winooski, VT) for analysis of plate data, which simplifies this process. 4. For samples which have been diluted, the TGFα concentration must be multiplied by the dilution factor (ie., if the sample was diluted five-fold, then the TGFα value obtained from the standard curve must be multiplied by five).
      Standard curve
      Sensitivity5 pg/ml
      Sensitivity NotesStudies have demonstrated that TGFα ELISA can distinguish 2.1 pg/ml from zero with a 95% confidence, therefor the ELISA can easly distinguish 6 pg/ml from zero.
      Assay Range0-1000 pg/ml
      Precision
      RecoveryHuman TGFα (900 pg/ml) was added to each of five (5) undiluted normal human sera (NHS) and five (5) undiluted normal human plasma (NHP). The spiked samples were analyzed neat (undiluted) and diluted 1:2, 1:4 and 1:8 with Assay Buffer. Results are expressed as a percent of the expected value +/- one standard deviation.
      Parallelism
      Specificity
      The TGFα ELISA detects soluble and intracellular TGFα. Specificity was demonstrated by immunoaffinity extraction (inhibition of assay signal) of TGFα positive samples by a specific TGFα antibody. The TGFα antibody, which is not a component of the ELISA, extracted almost all TGFα (almost the entire signal was lost), while the control antibody (non-TGFα antibody) did not affect the signal of the TGFα positive samples. The non-ELISA TGFα antibody extracts the same portion of the TGFα activity detected by this immunoassay from the biological samples as it did from a recombinant TGFα sample demonstrating that the assay is specificity for the soluble and intracellular TGFα.

      No measurable cross-reactivity was observed when human Epidermal Growth Factor (hEGF) and amphiregulin were assayed at a concentration greater of 1000 pg/ml.
      Registered TrademarksCalbiochem® is a registered trademark of Merck, KGaA, Darmstadt.
      Interactive Pathways™ is a trademark of Merck, KGaA, Darmstadt.