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QIA20 Cell Death Detection (Nuclear Matrix Protein) ELISA Kit

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Detection Methods: Colorimetric
QIA20
  
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      Übersicht

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      Designed for the quantitative detection of NMP41/7. The specific detection of soluble human NMPs afforded by the Cell Death Detection (Nuclear Matrix Protein) ELISA allows quantitation of cell death.
      Catalogue NumberQIA20
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Precision pipettors with disposable tips
      • Wash bottle or multichannel dispenser for washing
      • Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/595 nm.
      References
      ReferencesMiller, T.E., et al. 1994. In Current Communications in Cellular and Molecular Biology: Apoptosis II. 8, 357.
      Arguello, F., et al. 1993. Blood 82, 999a.
      Miller, T.E., et al. 1993. Biotechniques 15, 1042.
      Partin, A.W., et al. 1993. Cancer Res. 53, 744.
      Compton, D.A., et al. 1992. J. Cell Biol. 116, 1395.
      Miller, T.E., et al. 1992. Cancer Res. 52, 422.
      Yang, C.H., et al. 1992. J. Cell Biol. 116, 1303.
      Nakayasu, H. and R. Berezney. 1989. J. Cell Biol. 108, 1.
      Fey, E.G. and S. Penman. 1988. Proc. Natl. Acad. Sci. USA 85, 121.
      Zeitlin, S., et al. 1987. Mol. Cell Biol. 7, 111.
      Kumara-Siri, M.H. 1986. Biol. Chem. 261, 2844.
      Berrios, M., et al. 1985. Proc. Natl. Acad. Sci. USA 82, 4142.
      Pardoll, D.M., et al. 1980. Cell 19, 527.
      Berezney, R. and D.S. Coffey. 1974. Biochem. Biophys. Res. Commun. 60, 1410.
      Product Information
      Unit of DefinitionOne unit (U) is defined as the amount of NMP 41/7 released by 850 MCF7 cells maintained at a density of 1 - 5 x 10⁵ cells/ml in serum-free medium for 7 days.
      Detection methodColorimetric
      DeclarationUse of this product is covered by U.S. Patents 5,783,403, 5,882,876, 5,989,826, 6,162,607, 6,410,247, and 6,740,494. Foreign patents also cover the use of this product in numerous jurisdictions.
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Removable Strip Plate, Lyophilized NMP Standard, NMP Detector Antibody, Anti-FITC-POD, Substrate, ELISA Conjugant Diluent, 20X Plate Wash Concentrate, Stop Solution, Plate Sealer, and a user protocol.
      Applications
      Key Applications Enzyme-Linked Immunosorbent Assay
      Biological Information
      Assay range15-1000 U/ml
      Assay time4 h
      Sample TypeTissue culture supernatant
      Physicochemical Information
      Sensitivity10 U/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® Cell Death Detection (Nuclear Matrix Protein) ELISA Kit is used for quantitative determination of nuclear matrix protein 41/7, which is released from dead and dying cells. Nuclear matrix protein 41/7 appears to be the same as that designated as NuMA (nuclear mitotic apparatus protein) by some researchers.
      Storage and Shipping Information
      Ship Code Blue 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 arrival all components should be stored at -20°C. Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack. All of the reagents included in the Cell Death Detection (Nuclear Matrix Protein) ELISA have been tested for stability.
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Removable Strip Plate, Lyophilized NMP Standard, NMP Detector Antibody, Anti-FITC-POD, Substrate, ELISA Conjugant Diluent, 20X Plate Wash Concentrate, Stop Solution, Plate Sealer, and a user protocol.
      Specifications
      Global Trade Item Number
      Bestellnummer GTIN
      QIA20 0

      Documentation

      Cell Death Detection (Nuclear Matrix Protein) ELISA Kit Analysenzertifikate

      TitelChargennummer
      QIA20

      Literatur

      Übersicht
      Miller, T.E., et al. 1994. In Current Communications in Cellular and Molecular Biology: Apoptosis II. 8, 357.
      Arguello, F., et al. 1993. Blood 82, 999a.
      Miller, T.E., et al. 1993. Biotechniques 15, 1042.
      Partin, A.W., et al. 1993. Cancer Res. 53, 744.
      Compton, D.A., et al. 1992. J. Cell Biol. 116, 1395.
      Miller, T.E., et al. 1992. Cancer Res. 52, 422.
      Yang, C.H., et al. 1992. J. Cell Biol. 116, 1303.
      Nakayasu, H. and R. Berezney. 1989. J. Cell Biol. 108, 1.
      Fey, E.G. and S. Penman. 1988. Proc. Natl. Acad. Sci. USA 85, 121.
      Zeitlin, S., et al. 1987. Mol. Cell Biol. 7, 111.
      Kumara-Siri, M.H. 1986. Biol. Chem. 261, 2844.
      Berrios, M., et al. 1985. Proc. Natl. Acad. Sci. USA 82, 4142.
      Pardoll, D.M., et al. 1980. Cell 19, 527.
      Berezney, R. and D.S. Coffey. 1974. Biochem. Biophys. Res. Commun. 60, 1410.

      Broschüre

      Titel
      Caspases and other Apoptosis Related Tools Brochure

      Literaturstellen

      Titel
    • Sylvain Baize, et al. (1999) Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. Nature Medicine 5, 423-426.
      		•
      Anwenderprotokoll

      Revision18-June-2009 JSW
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival all components should be stored at -20°C. Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack. All of the reagents included in the Cell Death Detection (Nuclear Matrix Protein) ELISA have been tested for stability.
      Intended useThe Calbiochem® Cell Death Detection (Nuclear Matrix Protein) ELISA Kit is used for quantitative determination of nuclear matrix protein 41/7, which is released from dead and dying cells. Nuclear matrix protein 41/7 appears to be the same as that designated as NuMA (nuclear mitotic apparatus protein) by some researchers.
      BackgroundNuclear matrix proteins (NMPs) make up the internal structural framework of the nucleus and are associated with such functions as DNA replication, RNA synthesis, and hormone receptor binding. Work by Nakayasu and Berezney, Zeitlin et al., and Berrios et al. has indicated that NMPs are involved in regulation and coordination of gene expression. The identification of cell-type specific nuclear matrix proteins supports their potential contribution to cellular differentiation and tissue development. Although the nuclear matrix has been shown to be highly insoluble in vitro, it is now known that cell death releases soluble nuclear matrix proteins that can be detected in culture supernatant and other fluids containing dead and dying cells. The antibodies employed in the Cell Death Detection (Nuclear Matrix Protein) ELISA were developed to nuclear matrix proteins isolated from tissue culture cells by standard procedures. Both antibodies have been shown to react with lambda gt11 cDNA clones carrying published sequence from nuclear mitotic apparatus protein (NuMA), a nuclear matrix protein. More specifically, the Capture Antibody for NMP 41/7 reacts with a lambda gt11 cDNA clone encoding a peptide of approximately 60 kDa, located in the amino terminus of the published sequence for NuMA, and the FITC-anti-NMP 41/7 detector antibody reacts with a lambda gt11 cDNA clone encoding a peptide of ~96 kDa, located in the carboxy-terminus of the published sequence for NuMA. Because the level of NMP 41/7 detected in culture supernatant is a function of the number of dead and dying cells, the assay may be useful to quantify cell death. Other methods used to measure cell death include MTT, 51CR, trypan blue staining and ATP level. In comparison to these methods, the NMP 41/7 assay is advantageous since it measures antigen released directly from dying cells, therefore allowing differentiation between cytostatic and cytotoxic effects. Also, the NMP 41/7 assay requires only a small aliquot of material for testing, and does not require manual cell counts for quantification. In addition to measuring cell death in tissue culture, NMP 41/7 has been shown to be a useful marker for quantitative determination of engraftment, extension of disease and treatment response of human leukemia in SCID mice. The level of NMP 41/7 detected in SCID mouse serum correlates with human-derived tumor mass because the Cell Death Detection (Nuclear Matrix Protein) ELISA antibodies detect only human nuclear matrix protein. The antibodies have been shown to be reactive with a number of human cell lines; they may not react with all human cells. The antibodies have not been shown to be reactive with non-human cells. The user should make an initial determination of the assay's suitability for a particular use.
      Principles of the assayThe Calbiochem® Cell Death Detection (Nuclear Matrix Protein) ELISA Kit is a sandwich enzyme immunoassay employing mouse monoclonal antibodies for measurement of soluble nuclear matrix protein. A mouse monoclonal antibody specific for NMP 41/7 has been immobilized onto the surface of the plastic wells provided in the kit. The sample to be assayed is pipetted into the wells and allowed to incubate for one hour. NMP 41/7 contained in the samples or standards is captured during this incubation. Unbound material is washed away, and FITC-labeled detector monoclonal antibody added. The detector antibody also recognizes NMP 41/7, and will bind to any NMP 41/7 captured in the first incubation. After a second incubation and wash step, an HRP-linked sheep anti-FITC antibody is used to detect formation of NMP 41/7 antibody sandwiches. Un-reacted components are again removed by washing and a substrate solution is added to all wells, giving a colored end product. Results are read with an ELISA plate reader with the absorbance set at 450-595 nm. The intensity of the colored end product is proportional to the amount of soluble NMP 41/7 present in the original sample.
      Materials provided• Anti-NMP-Coated 96-Well Plate (Kit Component JA1531-1EA): 1 96-well plate, coated with mouse anti-NMP 41/7, supplied as six 2 x 8-well strips
      • Lyophilized NMP Standard (Kit Component No. JA9410-EA): 1 vial, please refer to vial label for reconstitution information
      • Assay Diluent (Kit Component No. JA7644-50ML): 1 bottle, 50 ml
      • NMP Detector Antibody, 1X (Kit Component No. JA9411-12ML): 1 vial, 12 ml
      • Anti-FITC-POD, supplied as 200X (Kit Component No. JA9412-100UL ): 1 vial, 100 µl
      • ELISA Conjugate Diluent, 1X (JA1615-12ML): 1 bottle, 12 ml
      • ELISA 20X Plate Wash Concentrate (Kit Component JA1617-100ML): 1 bottle, 100 ml
      • TMB Substrate (Kit Component JA1608-12ML): 1 vial, 12 ml, ready-to-use
      • ELISA Stop Solution (Kit Component JA1616-12ML): 1 bottle, 12 ml, 2.5 N H2SO4
      • Plate Sealers: 2 each
      Materials Required but not provided• Precision pipettors with disposable tips
      • Wash bottle or multichannel dispenser for washing
      • Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/595 nm.
      PreparationRemove medium from cells and centrifuge at 2000 rpm for 10 min and collect the supernatant. At this point, the sample may be used undiluted in the assay or frozen at -20°C for later use. The assay is also suitable for measuring total cellular NMP derived from cell lysates.
      Reagent preparationAllow kit components and samples to reach room temperature prior to carrying out the assay. • ELISA 1X Plate Wash: Prepare ELISA 1X Plate Wash by adding 25 ml of the ELISA 20X Plate Wash Concentrate to 475 ml of deionized water. Mix well. • 1X Anti-FITC-POD: Prepare 1X Anti-FITC-POD Conjugate Solution by adding 60 µl Anti-FITC-POD 200X to 11.94 ml ELISA Conjugate Diluent. Mix well. • NMP Standard: Reconstitute the Lyophilized NMP Standard with the Assay Diluent as indicated on the vial label. Prepare a standard curve by making dilutions of the reconstituted Lyophilized NMP Standard in Assay Diluent as indicated in the table below. Unused standard can be dispensed into aliquots and stored at -20°C for further use.

      Table 1: Standard Dilutions
      Detailed protocol1. Removed the desired number of strips from the Anti-NMP-Coated 96-Well Plate and place them in the frame. Return unused strips to the pouch and re-seal the edge with tape. Store unused strips at 4°C.
      2. Add 100 µl of diluted standards and samples to designated wells. Cover with a Plate Sealer and incubate for 2 h at room temperature.
      3. Wash the plate by completely filling the wells with ELISA 1X Plate Wash; shake the contents of the wells into the sink and remove excess liquid by tapping the inverted plate on paper towels. Repeat for a total of 3 washes.
      4. Add 100 µl NMP Detector Antibody to each well. Cover with a Plate Sealer and incubate for 1 h at room temperature.
      5. Wash the plate as indicated in step 4.
      6. Add 100 µl 1X Anti-FITC-POD to each well. Cover with a Plate Sealer and incubate for 1 h at room temperature.
      7. Wash the plate as indicated in step 4.
      8. Add 100 µl TMB Substrate to each well and incubate for 30 min at room temperature in the dark.
      9. Add 100 µl ELISA Stop Solution and read the assay at a dual wavelength of 450/595 nm (550 nm and 540 nm can be used as alternative wavelengths to 595 nm. If a dual wavelength reader not available, read only at 450 nm).
      CalculationsSeveral options are available for the calculation of the concentration of NMP in the samples. We recommend the use of an immunoassay software package capable of generating a four parameter logistic (4-PL) curve-fit. If data reduction software is not available, construct a standard curve by plotting the mean absorbance for each standard on the Y-axis against the protein concentration on the X-axis and draw a best-fit curve through the points on the graph. Values are expressed in units per milliliter (U/ml).
      Standard curve

      Figure 1: Standard Curve

      The NMP standard was reconstituted, diluted, and assayed as outlined in the Detailed Protocol.
      Limitations of the assayAny sample with an absorbance above the highest standard should be interpreted as containing more NMP 41/7 than the highest standard. Dilute these samples with Assay Diluent and reassay. The concentration of NMP 41/7 in the original sample would be determined by multiplying the result obtained from the standard curve by the dilution factor.
      Sensitivity10 U/ml
      Assay Range15-1000 U/ml
      SpecificityThe Calbiochem® Cell Death Detection (Nuclear Matrix Protein) ELISA Kit is specific for human nuclear matrix protein 41. The antibodies used in this kit are not known to cross react with non-human proteins.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.