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CBA078 PhosphoDetect™ Hsp27 (pSer78/82) ELISA Kit

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Detection Methods: Colorimetric
CBA078
  
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      Übersicht

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      OverviewDetects and quantifies the level of phosphorylated Hsp27 protein in cell lysates, tissue extracts, and biological fluids. Hsp27 is the small heat shock protein that acts as a molecular chaperone interacting with a large number of proteins. Hsp27 is known for its ability to negatively regulate the mitochondrial pathway to caspase activation. The phosphorylated form of Hsp27 also plays a major role in apoptosis through its ablity to prevent cell death by maintaining redox homeostasis and mitochondrial stability.
      Catalogue NumberCBA078
      Brand Family Calbiochem®
      Application Data
      A standard curve was generated using phosphorylated (black square) and unphosphorylated (black triangle) Hsp27 as outlined in the Detailed Protocol. Assay Range: 0.1-10 ng/ml; lower limit of detection: ~0.1 ng/ml.

      Various tumor cell lines (indicated in the figure) were exposed to 100 J/m2 UV irradiation or treated with 400 mM sorbitol for 30 min. Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the recommended protocol. The level of phosphorylated Hsp27 was determined as outlined in the Detailed Protocol and expressed as ng phosphorylated Hsp27/mg total protein.

      (A) MCF-7 and HeLa cells were either left untreated (-UV) or exposed to UV-C radiation (+UV). Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009), subjected to SDS-PAGE, and transferred to nitrocellulose membrane for Western blotting analysis using Anti-Phospho-Hsp27 Detector Antibody Lane 1: MWM; lane 2: MCF-7, -UV; lane 3: MCF-7, +UV; lane 4: HeLa, -UV; lane 5: HeLa, +UV. (B) Unphosphorylated Hsp27 and recombinant Hsp27 that had been phosphorylated in vitro with MAPKAP-2 kinase were subjected to SDS-PAGE and Western blotting analysis using Anti-Phospho-Hsp27 Detector Antibody. Lane 1: MWM; lane 2: in vitro phosphorylated, recombinant Hsp27; lane 3: unphosphorylated, recombinant Hsp27.

      MCF7 cells were either left untreated (left bar) or treated with a highly specific, cell-permeable inhibitor of p38 MAP kinase, SB203580 (Cat. No. 559389), (2 µM) followed by UV treatment (right bar). Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) and the level of phosphorylated Hsp27 was determined as outlined in the Detailed Protocol.
      Materials Required but Not Delivered• Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm.
      • Wash bottle or multi-channel dispenser for washing
      References
      ReferencesXu, L., et al. 2006. Oncogene 25, 2987.
      Shin, K.D., et al. 2005. J. Biol. Chem. 280, 41439.
      Whitesell, L. and Lindquist, S. I. 2005. Nature Rev. 5, 761.
      Eustace, B.K., et al. 2004. Nature Cell Biol. 6, 507.
      Bruey, J-M., et al. 2000. Nature Cell Biol. 2, 645.
      Cornford, P.A., et al. 2000. Cancer Res. 60, 7099.
      Garrido, C., et al. 1999. FASEB J. 13, 2061.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsHsp27-Coated Plate, Rabbit Anti-Hsp27 Detector Antibody, Phosphorylated Hsp27 Standard, HRP-Conjugate, Assay Diluent, ELISA 20X Plate Wash Concentrate, TMB Substrate, ELISA Stop Solution, and a user protocol
      Positive controlPhosphorylated Hsp27 Standard, UV-treated HeLa or MCF7 cells or sorbitol-treated DU145 cells
      Applications
      Biological Information
      Assay range0.1-10 ng/ml
      Assay time4.5 h
      Sample TypeCell lysates
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® PhosphoDetect™ Hsp27 (pSer78/82) ELISA Kit is intended for the quantitative measurement of Hsp27 that is phosphorylated at Ser⁷⁸ and Ser⁸². The assay has been validated with human cell lysates and does not detect unphosphorylated Hsp27.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Regulatory Review
      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 store the entire contents of the kit at -20°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsHsp27-Coated Plate, Rabbit Anti-Hsp27 Detector Antibody, Phosphorylated Hsp27 Standard, HRP-Conjugate, Assay Diluent, ELISA 20X Plate Wash Concentrate, TMB Substrate, ELISA Stop Solution, and a user protocol
      Specifications
      Global Trade Item Number
      Bestellnummer GTIN
      CBA078 0

      Documentation

      PhosphoDetect™ Hsp27 (pSer78/82) ELISA Kit Analysenzertifikate

      TitelChargennummer
      CBA078

      Literatur

      Übersicht
      Xu, L., et al. 2006. Oncogene 25, 2987.
      Shin, K.D., et al. 2005. J. Biol. Chem. 280, 41439.
      Whitesell, L. and Lindquist, S. I. 2005. Nature Rev. 5, 761.
      Eustace, B.K., et al. 2004. Nature Cell Biol. 6, 507.
      Bruey, J-M., et al. 2000. Nature Cell Biol. 2, 645.
      Cornford, P.A., et al. 2000. Cancer Res. 60, 7099.
      Garrido, C., et al. 1999. FASEB J. 13, 2061.
      Anwenderprotokoll

      Revision13-March-2008 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      StorageUpon arrival store the entire contents of the kit at -20°C.
      Intended useThe Calbiochem® PhosphoDetect™ Hsp27 (pSer78/82) ELISA Kit is intended for the quantitative measurement of Hsp27 that is phosphorylated at Ser⁷⁸ and Ser⁸². The assay has been validated with human cell lysates and does not detect unphosphorylated Hsp27.
      BackgroundThe small heat shock protein, Hsp27, is expressed at various levels in different cell types and tissues, as well as during different stages of development and differentiation. It is regulated at both the transcriptional and post-translational levels. Hsp27 levels increase several-fold over basal levels under specific, stressful, environmental conditions that are known to activate this heat shock transcription factor. This overexpression confers cellular resistance to a variety of stimuli that induce cell death with either necrotic or apoptotic events. Hsp27 is also regulated at the posttranslational level by stress or growth factors/cytokines that activate stress-activated protein kinase 2 (SAPK2) p38, the upstream activator of the Hsp27 kinase mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2. Hsp27 is a molecular chaperone that can interact with a large number of proteins. Recent evidence has shown that Hsp27 regulates apoptosis through its interaction with key components of the apoptotic signaling pathway, in particular, those involved in caspase activation and apoptosis. Thus, Hsp27 functions as an anti-apoptotic molecule. In the intrinsic pathway of apoptosis, Hsp27 prevents the formation of the apoptosome and subsequent activation of caspases by directly sequestering cytochrome c. Hsp27 has also been shown to interact with caspase-3. The phosphorylated from of Hsp27 has been shown to directly interact with Daxx to prevent cell death. The mechanism by which Hsp27 exerts its protective effects may be a result of its role in maintaining redox homeostasis and mitochondrial stability. This negative regulation of the mitochondrial pathway and the blocking of caspase activation by interacting with cytochrome c may represent an important aspect of the physiological role of Hsp27.
      Principles of the assayThe Calbiochem® PhosphoDetect™ Hsp27 (pSer78/82) ELISA Kit is a quantitative sandwich immunoassay for the quantitation of phosphorylated Hsp27 in human cell lysates, tissue extracts, and biological fluids. The assay utilizes an Hsp27-specific monoclonal antibody, immobilized on the wells of a 96-well plate, to capture Hsp27. Unbound material is washed from the wells and a phospho-Hsp27 (pSer78/82) rabbit, polyclonal antibody is used to detect phosphorylated Hsp27. A goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) is used as a secondary antibody, followed by the addition of the substrate. When HRP acts on the substrate, a blue color develops. Sensitivity is increased by the addition of a sulfuric acid stop solution, which yields a yellow color. The absorbance is measuring at 450 nm (preferably with reference wavelength set at 540-600 nm). The level of Hsp27 is calculated using a standard curve.
      Materials provided• Anti-Hsp27 Antibody Coated 96-Well Plate (Kit Component No. JA7650-1EA): 1 plate, 96 wells, supplied as six 2 x 8-well strips
      • Phosphorylated Hsp27 Standard (Kit Component No. JA9368-20NG): 2 vials, 20 ng lyophilized, phosphorylated, recombinant Hsp27
      • Rabbit Anti-Phospho-Hsp27 Detector Antibody (Kit Component No. JA9367-120UL): 1 vial, 120 µl phospho-Hsp27 (pSer78/82) antibody supplied as 100X
      • HRP Conjugate (Kit Component No. JA7922-100UL): 1 vial, 100 µl goat anti-rabbit IgG conjugated to HRP, supplied as 200X
      • Assay Diluent (Kit Component No. JA7644-50ML): 1 bottle, 50 ml
      • ELISA 20X Plate Wash Concentrate (Kit Component No. JA1617-100ML): 1 bottle, 100 ml
      • TMB Substrate (Kit Component No. JA1608-12ML): 1 bottle, 12 ml, ready to use
      • ELISA Stop Solution (Kit Component No. JA1616-12ML): 1 bottle, 12 ml, 2.5 N H₂SO₄ ready to use
      • Plate Sealers: 2 each
      Materials Required but not provided• Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm.
      • Wash bottle or multi-channel dispenser for washing
      Reagent preparationAllow kit components and samples to reach room temperature (18-25°C). • 1X ELISA Plate Wash: Prepare 1X ELISA Plate Wash by adding 25 ml ELISA 20X Plate Wash Concentrate to 475 ml of deionized water. Mix well. • 1X Rabbit Anti-Phospho-Hsp27 Detector Antibody: Prepare a 1X Rabbit Anti-Hsp27 Detector Antibody by diluting the Rabbit Anti-Hsp27 Detector Antibody 1:100 with Assay Diluent; mix well. For example, to prepare enough Rabbit Anti-Phospho-Hsp27 Detector Antibody for the two 8-well strips, add 20 µl Rabbit Anti-Hsp27 Detector Antibody to 1980 µl Assay Diluent. • 1X HRP-Conjugate: Prepare 1X HRP-Conjugate by diluting the HRP-Conjugate 1:200 with Assay Diluent; mix well. For example, to prepare enough HRP-Conjugate 1X for two 8-well strips, add 10 µl HRP-Conjugate to 1990 µl Assay Diluent. • Phosphorylated Hsp27 Standard: Reconstitute a vial of Phosphorylated Hsp27 Standard with 2 ml diH2O to yield a stock solution of 10 ng/ml. Unused standard can be dispensed into aliquots and stored at -70°C for future use. Prepare a series of standard dilutions as outlined in the table below; these diluted standards will be used to create a standard curve for the assay:

      Table 1: Standard Curve Dilutions
      Detailed protocol1. Remove the desired number of strips from the Anti-Hsp27 Coated 96-Well Plate and place them in the frame. Return the unused strips to the foil pouch and reseal the entire edge with tape. Store the unused strips at 4°C.
      2. Add 100 µl diluted standards and samples to designated wells. Cover the wells with a Plate Sealer and incubate for 90 min at room temperature.
      3. Wash the wells by completely filling each well with 1X ELISA Plate Wash. Shake the contents into the sink and tap the inverted plate on paper towels to remove excess liquid. The complete removal of liquid at each step is essential for good assay performance. Repeat for a total of 3 washes. Following the last wash, remove any remaining liquid by thoroughly aspirating or decanting and tapping the plate on paper towels.
      4. Add 100 µl 1X Rabbit Anti-Hsp27 Detector Antibody to each well. Cover the wells with a Plate Sealer and incubate for 90 min at room temperature.
      5. Wash the plate as indicated in step 3.
      6. Add 100 µl 1X HRP-Conjugate to each well. Cover the wells with a Plate Sealer and incubate for 60 min at room temperature.
      7. Wash the plate as indicated in step 3.
      8. Add 100 µl TMB Substrate to each well and incubate for 10-20 min at room temperature.
      9. Add 100 µl ELISA Stop Solution to each well and measure the absorbance 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 option is not available, read only at 450 nm).
      CalculationsSeveral options are available for the calculation of the concentration of phosphorylated Hsp27 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 of the X-axis and draw a best-fit curve through the points on the graph. Values are expressed in ng/ml.
      Standard curve

      Figure 1: Representative Standard Curve

      A standard curve was generated using phosphorylated (black square) and unphosphorylated (black triangle) Hsp27 as outlined in the Detailed Protocol. Assay Range: 0.1-10 ng/ml; lower limit of detection: ~0.1 ng/ml.
      Example data

      Figure 2: Quantification of Phosphorylated Hsp27 in Tumor Cells

      Various tumor cell lines (indicated in the figure) were exposed to 100 J/m2 UV irradiation or treated with 400 mM sorbitol for 30 min. Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the recommended protocol. The level of phosphorylated Hsp27 was determined as outlined in the Detailed Protocol and expressed as ng phosphorylated Hsp27/mg total protein.

      Figure 3: Detection of Phosphorylated Hsp27 by Western Blotting

      (A) MCF-7 and HeLa cells were either left untreated (-UV) or exposed to UV-C radiation (+UV). Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009), subjected to SDS-PAGE, and transferred to nitrocellulose membrane for Western blotting analysis using Anti-Phospho-Hsp27 Detector Antibody Lane 1: MWM; lane 2: MCF-7, -UV; lane 3: MCF-7, +UV; lane 4: HeLa, -UV; lane 5: HeLa, +UV. (B) Unphosphorylated Hsp27 and recombinant Hsp27 that had been phosphorylated in vitro with MAPKAP-2 kinase were subjected to SDS-PAGE and Western blotting analysis using Anti-Phospho-Hsp27 Detector Antibody. Lane 1: MWM; lane 2: in vitro phosphorylated, recombinant Hsp27; lane 3: unphosphorylated, recombinant Hsp27.

      Figure 4: Phosphorylation of Hsp27 in the Presence of Inhibitor

      MCF7 cells were either left untreated (left bar) or treated with a highly specific, cell-permeable inhibitor of p38 MAP kinase, SB203580 (Cat. No. 559389), (2 µM) followed by UV treatment (right bar). Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) and the level of phosphorylated Hsp27 was determined as outlined in the Detailed Protocol.
      Assay Range0.1-10 ng/ml
      Protocol Summary

      Figure 5: Protocol Summary
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      PhosphoDetect™, CytoBuster™, and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.