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QIA26 Fas/APO-1 ELISA Kit

Fas/APO-1 ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
QIA26
  
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      Overview

      Replacement Information
      Description
      OverviewRapid, precise assay for the in vitro quantitation of human Fas/APO-1 protein. Measures both membrane bound and soluble Fas protein.
      Catalogue NumberQIA26
      Brand Family Calbiochem®
      Application Data
      Materials Required but Not Delivered• Microplate reader capable of measuring absorbance at 450 nm • Precision pipettes that deliver 2 µl to 1 ml volumes • Adjustable 1-25 ml pipettes for reagent preparation • 100 ml and 1 liter graduated cylinders • Absorbent paper • Distilled or deionized water • Log-log graph paper or computer and software for ELISA data analysis • Tubes to prepare standard or sample dilutions • Protease Inhibitor Cocktail Set III (Cat. No. 539134)
      References
      ReferencesNagata, S. and Goldstein, P. 1995. Science 267, 1449. Nagata, S. 1994. Adv. Immunol. 57, 129. Mapara, M.V., et al. 1993. Eur. J. Immunol. 23, 702.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit contains96-well coated plate, Fas standard, assay diluent, biotinylated anti-Fas detector antibody, HRP-streptavidin concentrate, lysate buffer, TMB substrate, stop solution, wash concentrate, and a user protocol.
      Applications
      Biological Information
      Assay time4.75 h
      Sample Typehuman tissue lysate, cell lysate
      Physicochemical Information
      Sensitivity> 0.8 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem Fas/APO-1 Assay is a non-isotopic immunoassay for the in vitro quantitation of human Fas/APO-1/CD95 proteins in cell lysates and tissue extracts.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Regulatory Review
      Storage Multiple storage requirements
      Storage ConditionsUpon arrival store the Fas Standard at -20°C and the remaining components of the kit at 4°C. Following reconstitution of the Fas Standard, aliquot and freeze (-70°C).
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit contains96-well coated plate, Fas standard, assay diluent, biotinylated anti-Fas detector antibody, HRP-streptavidin concentrate, lysate buffer, TMB substrate, stop solution, wash concentrate, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA26 0

      Documentation

      References

      Reference overview
      Nagata, S. and Goldstein, P. 1995. Science 267, 1449. Nagata, S. 1994. Adv. Immunol. 57, 129. Mapara, M.V., et al. 1993. Eur. J. Immunol. 23, 702.
      User Protocol

      Revision12-September-2007 JSW
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the Fas Standard at -20°C and the remaining components of the kit at 4°C. Following reconstitution of the Fas Standard, aliquot and freeze (-70°C).
      Intended useThe Calbiochem Fas/APO-1 Assay is a non-isotopic immunoassay for the in vitro quantitation of human Fas/APO-1/CD95 proteins in cell lysates and tissue extracts.
      BackgroundFas (APO-1 or CD95) is a cell-surface receptor that transduces apoptotic signals from Fas ligand (FasL). Fas and FasL belong to the TNF superfamily and are type I and type II transmembrane proteins, respectively. Fas and FasL have been observed as soluble molecules in addition to their membrane-associated forms. Fas is expressed to a large extent on activated T and B lymphocytes, and on malignant lymphoid cells.
      Principles of the assayThe Calbiochem® Fas/APO-1 Assay is a sandwich ELISA for the detection and quantitation of human Fas. An antibody, specific for the human Fas protein, has been immobilized on the surface of the wells of a 96-Well Plate. The sample to be assayed is pipetted into the wells and any Fas present will bind to the capture antibody. Unbound material is washed away and biotinylated detector monoclonal antibody is added. The detector antibody also recognizes human Fas protein, and will bind to any Fas that has been retained by the capture antibody. The detector antibody, in turn, is bound by horseradish peroxidase-conjugated streptavidin. The horseradish peroxidase catalyzes the conversion of the chromogenic substrate, tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of Stop Solution), the intensity of which is proportional to the amount of Fas protein in the sample. The colored reaction product is quantified using a spectrophotometer. Quantitation is achieved by the construction of a standard curve using known concentrations of Fas. By comparing the absorbance obtained from a sample containing an unknown amount of Fas with that obtained from the standards, the concentration of Fas in the sample can be determined.
      Materials provided• FAS/APO Coated ELISA Plate (Kit Component No. JA9415): 1 plate, 96-wells, supplied as twelve 8-well strips • Fas Standard (Kit Component No. JA9416): 2 vials, lyophlized recombinant human Fas • Fas/APO Detector Antibody (Kit Component No. JA9418): 2 vials, lyophilized, biotinylated anti-Fas antibody • Streptavidin-HRP (Kit Component No. JA9419): 1 vial, 8 µl, supplied as 32000X • Assay Diluent (Kit Component No. JA9417): 1 vial, 15 ml, supplied as 5X • Lysate Buffer (Kit Component No. JA9420): 1 vial, 5 ml, supplied as 2X • TMB Substrate (Kit Component No. JA9421): 1 vial, 12 ml, ready-to-use • Stop Solution (Kit Component No. JA9422): 1 vial, 8 ml, ready-to-use • Wash Concentrate (Kit Component No. JA9423): 1 bottle, 25 ml, supplied as 20X
      Materials Required but not provided• Microplate reader capable of measuring absorbance at 450 nm • Precision pipettes that deliver 2 µl to 1 ml volumes • Adjustable 1-25 ml pipettes for reagent preparation • 100 ml and 1 liter graduated cylinders • Absorbent paper • Distilled or deionized water • Log-log graph paper or computer and software for ELISA data analysis • Tubes to prepare standard or sample dilutions • Protease Inhibitor Cocktail Set III (Cat. No. 539134)
      Preparation• Preparation of Cell Lysates and Tissue Extracts: Cell lysates and tissue extracts can be prepared using conventional methods, including glass-bead "smash", douncing, freeze-thaw, mortar and pestle, or a combination of any of these methods. The method of choice may depend on the nature of the protein, so it may be necessary to research published literature for information on the optimal method for a given protein(s). For convenience, the kit is provided with a lysis buffer (Lysate Buffer, JA9420) that is compatible with the assay. The following guidelines may be used to prepare cell lysates and tissue extracts for the assay: 1. Add 500 µl Lysate Buffer per 1 x 106 cells or 10 mg tissue. Different cell and tissue types will contain different amounts of total protein, so the volume of Lysate Buffer should be adjusted as necessary for individual sample types. The total protein concentration should be ≥1 mg/ml after homogenization. 2. Homogenize the cells or tissue using the method of choice. 3. Centrifuge the samples at 10,000 x g for 1 min or 5000 x g for 2 min to pellet the debris. Transfer the supernatant to a clean tube. 4. Determine the protein concentration using the method of choice. Adjust the concentration so that the same concentration of protein is added for each sample. Aliquot and store unused sample at -70°C. • Dilution of Samples: Cell lysates and tissue extracts should be diluted at least 5-fold with 1X Assay Diluent prior to performing the assay.
      Reagent preparationNote: Warm all reagents and samples to room temperature (18-25°C) prior to use. • 1X Assay Diluent: Dilute the Assay Diluent 5-fold with deionized or distilled water. • Fas Standard: Briefly centrifuge the vial of Fas Standard to pellet the contents. Add 100 µl 1X Assay Diluent to the vial and mix gently to reconstitute. The stock concentration is 1 µg/ml. To prepare serial dilutions for a standard curve, label a series of tubes 1 (200), 2 (66.67), 3 (22.22), 4 (7.41), 5 (2.47), 6 (0.823), and 7 (0). Add 320 µl 1X Assay Diluent to tube 1 and 300 µl 1X Assay Diluent to tubes 2-7. Add 80 µl reconstituted Fas Standard to tube 1 and mix thoroughly; the resulting concentration is 200 ng/ml. Using a fresh pipette tip, transfer 150 µl from tube 1 to tube 2 and mix thoroughly. Using a fresh pipette tip, transfer 150 µl from tube 2 to tube 3 and mix thoroughly. Continue transferring 150 µl as above (see figure below) until you reach tube 6; do not transfer any liquid to tube 7, as this will serve as the 0 ng/ml standard.

      Figure 1: Preparation of Standard Curve

      • 1X Wash Buffer: If the Wash Concentrate contains visible crystals, warm to room temperature and mix gently until redissolved. Dilute 20 ml Wash Buffer Concentrate with 380 ml deionized or distilled water to yield 400 ml 1X Wash Buffer. • Fas/APO Detector Antibody Stock: Briefly centrifuge the vial of Fas/APO Detector Antibody to pellet the lyophilized material. Add 100 µl 1X Assay Diluent to the vial and mix gently to reconstitute. The Fas/APO Detector Antibody Stock can be stored at 4°C for 5 days. Dilute this stock 80-fold with 1X Assay Diluent for use in the assay. • 1X Streptavidin-HRP: Briefly centrifuge the vial of Streptavidin-HRP. Dilute the Streptavidin-HRP 32,000-fold with 1X Assay Diluent. • 1X Lysate Buffer: Dilute the Lysate Buffer 2-fold with deionized or distilled water prior to preparation of cell lysates or tissue extracts. It is recommended that protease inhibitor cocktail be added to the 1X Lysate Buffer prior to homogenization of cells/tissue (see Sample Preparation).
      Detailed protocolNote: It is recommended that all standards and samples be run in duplicate. 1. Add 100 µl of each standard and sample to designated wells. Cover the wells and incubate for 2.5 h at room temperature or overnight at 4°C. 2. Discard the contents of the wells and wash the plate by adding 200 µl 1X Wash Buffer to all wells and incubating for 1 min. Discard the 1X Wash Buffer and repeat this for a total of 4 washes. Thoroughly discard the contents following the final wash by tapping the inverted plate on absorbent papter. 3. Add 100 µl 1X Fas/APO Detector Antibody to each well. Incubate for 1 h at room temperature. 4. Discard the contents of the wells and wash 4 times as outlined in step 2. 5. Add 100 µl 1X Streptavidin-HRP to each well. Incubate for 45 min at room temperature. 6. Discard the contents of the wells and wash 5 times as outlined in step 2. 7. Add 100µl TMB One-Step Substrate to each well. Incubate for 30 min at room temperature in the dark. 8. Add 50 µl Stop Solution to each well. Read at 450 nm immediately.
      CalculationsCalculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard absorbance. 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.
      Standard curveThis standard curve represents a typical standard curve. However a standard curve must be run with each assay.

      Figure 2: Standard Curve
      Sensitivity> 0.8 ng/ml
      Sensitivity NotesThe minimum detectable level of Fas is typically less than 0.8 ng/ml.
      RecoveryRecovery was determined by spiking various levels of human Fas into human tissue extracts and cell lysates. Mean recoveries are as follows:

      Table 1: Fas Recovery
      ReproducibilityIntra-assay: CV <10% Inter-assay: CV <12%
      Linearity

      Table 2: Linearity of the Assay
      SpecificityCross Reactivity: This assay shows no cross-reactivity with any of the cytokines tested (i.e., human Angiogenin, BDNF, BLC, ENA-78, FGF-4, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, IL-309, IP-10, G-CSF, GM-CSF, IFN-γ, Leptin, MCP-1, MCP-2, MCP-3, MDC, MIP-1α, MIP-1 β, MIP-1d, PARC, PDGF, RANTES, SCF, TARC, TGF-β, TIMP-1, TIMP-2, TNF-α, TNF-β, TPO, VEGF).
      Protocol Summary

      Figure 3: Protocol Summary
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.