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QIA119 Hsp27 ELISA Kit

Hsp27 ELISA Kit : FDS (Fiches de données de sécurité), certificats d’analyse (CoA) et de qualité (CoQ), dossiers, brochures et autres documents disponibles.

Synonymes: Heat Shock Protein 27 ELISA Kit

Detection Methods: Colorimetric
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      Aperçu

      Replacement Information

      Tableau de caractéristiques principal

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      Hsp27 over-expression has been reported to be a negative regulator of apoptosis. Elevated levels of Hsp27 may be associated with aggressive growth and cell invasion, which often results in poor prognosis in breast, ovarian, and prostatic carcinomas. In neuronal cells, Hsp27 over-expression promotes survival following growth factor withdrawal or other stress.
      Catalogue NumberQIA119
      Brand Family Calbiochem®
      SynonymsHeat Shock Protein 27 ELISA Kit
      Materials Required but Not Delivered• Precision pipettors with disposable tips
      • Wash bottle or multichannel dispenser for washing
      • PBS
      • Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/540 nm or 450/595 nm
      References
      ReferencesCornford, P.A., 2000. Cancer Res. 60, 7099.
      Arts, H.J., 1999. Int. J. Cancer 84, 234.
      Garrido, C., et al. 1997. Cancer Res. 57, 2661.
      Mehlen, P., et al. 1997. J. Biol. Chem. 272, 31657.
      Mairesse, N., et al. 1996. Cell Biol. Int. 20, 205.
      Minowada, G., and Welch, W. 1995. J. Biol. Chem. 270, 7047.
      Piotrowicz, R.S. 1995. FASEB J. 9, 1079.
      Rouse, J., et al. 1994. Cell 78, 1027.
      Oesterreich, S., et al. 1993. Cancer Res. 53, 4443.
      Landry, J., et al. 1991. J. Biol. Chem. 267, 794.
      Thor, A., et al. 1991. J. Natl. Cancer Inst. 83, 170.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsAnti-Hsp27 Coated 96-Well Plate, Hsp27 Standard, Lysis Buffer, Assay Diluent, Detector Antibody, Wash Buffer, HRP-Conjugate, TMB, Stop Solution, and a user protocol.
      Applications
      Key Applications Enzyme-Linked Immunosorbent Assay
      Biological Information
      Assay range0.02-1 ng/ml
      Assay time3.5 h
      Sample TypeCell lysates, plasma, serum, tissue extracts
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® Hsp27 ELISA Kit is intended for the quantitative determination of human Hsp27 in cell lysates, tissue extracts, serum, and plasma.
      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 arrival store the entire contents of the kit at -20°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAnti-Hsp27 Coated 96-Well Plate, Hsp27 Standard, Lysis Buffer, Assay Diluent, Detector Antibody, Wash Buffer, HRP-Conjugate, TMB, Stop Solution, and a user protocol.
      Specifications
      Global Trade Item Number
      Référence GTIN
      QIA119 0

      Documentation

      Hsp27 ELISA Kit Certificats d'analyse

      TitreNuméro de lot
      QIA119

      Références bibliographiques

      Aperçu de la référence bibliographique
      Cornford, P.A., 2000. Cancer Res. 60, 7099.
      Arts, H.J., 1999. Int. J. Cancer 84, 234.
      Garrido, C., et al. 1997. Cancer Res. 57, 2661.
      Mehlen, P., et al. 1997. J. Biol. Chem. 272, 31657.
      Mairesse, N., et al. 1996. Cell Biol. Int. 20, 205.
      Minowada, G., and Welch, W. 1995. J. Biol. Chem. 270, 7047.
      Piotrowicz, R.S. 1995. FASEB J. 9, 1079.
      Rouse, J., et al. 1994. Cell 78, 1027.
      Oesterreich, S., et al. 1993. Cancer Res. 53, 4443.
      Landry, J., et al. 1991. J. Biol. Chem. 267, 794.
      Thor, A., et al. 1991. J. Natl. Cancer Inst. 83, 170.
      Protocole Utilisateur

      Revision13-August-2008 JSW
      SynonymsHeat Shock Protein 27 ELISA Kit
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at -20°C.
      Intended useThe Calbiochem® Hsp27 ELISA Kit is intended for the quantitative determination of human Hsp27 in cell lysates, tissue extracts, serum, and plasma.
      BackgroundThe small heat shock protein Hsp27 is involved in thermotolerance, as well as in other cellular functions such as signal transduction, cell differentiation, and cell proliferation. An increasing amount of evidence indicates that Hsp27 may also play a role in cell growth and motility. High levels of Hsp27 may be associated with aggressive cell growth and cell invasion. Elevated levels of Hsp27 have been linked to poor prognoses for breast, ovarian, and prostatic carcinomas. Phosphorylation occurring at serine residues 15, 78, and 82 is a key regulator of Hsp27 function. Hsp27 inhibits apoptotic pathways triggered by a variety of stimuli in mammalian cells. It inhibits the activation of procaspase-9 by cytochrome c released in the cytosol, thereby inhibiting the activation of downstream procaspases such as procaspase 3. It has been shown that the inhibition of Hsp27 accumulation during the early differentiation stage of murine embryonic stem cells is sufficient to abort the differentiation program because of the overall death of the cells by apoptosis. Hsp27 may therefore represent the first example of a ubiquitously induced anti-apoptotic protein present during early cell differentiation.
      Principles of the assayThe Calbiochem® Hsp27 ELISA Kit is a complete kit for the quantitative determination of human Hsp27 in cell lysates, tissue extracts, and biological fluids such as serum or plasma. A mouse anti-human Hsp27 monoclonal antibody is pre-coated on the wells of a 96-well plate and serves as the capture antibody. Hsp27 standard dilutions and samples, together with a rabbit polyclonal detection antibody specific for human Hsp27, are simultaneously incubated in the pre-coated wells. Following a wash step a goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate is added, which binds to the polyclonal human Hsp27 antibody. Substrate is added and its subsequent reaction with the HRP results in a blue-colored solution. Sensitivity is increased by the addition of a sulfuric acid stop solution, yielding a yellow color. The absorbance is read at 450 nm and Hsp27 levels are determined by comparing the absorbance of samples with the values obtained from the standard curve.
      Materials provided• Anti-Hsp27 Antibody Coated 96-Well Plate (Kit Component No. JA7650-1EA): 1 plate, 96 wells, supplied as eight 12-well strips, pre-coated with anti-Hsp27 mouse monoclonal antibody
      • Hsp27 Standard (Kit Component No. JA7651-1EA): 2 vials, 250 ng each, lyophilized human recombinant Hsp27
      • Hsp27 Detection Antibody (Kit Component No. JA7652-150UL): 1 vial, 150 µl anti-human Hsp27 polyclonal antibody, supplied as 500X
      • HRP Conjugate (Kit Component No. JA7653-100UL): 1 vial, 100 µl anti-rabbit IgG HRP conjugate, supplied as 500X
      • Assay Buffer (Kit Component No. JA7644-50ML): 1 bottle, 50 ml, buffered saline containing proteins
      • Wash Buffer Concentrate (Kit Component No. JA1617-100ML): 1 bottle, 100 ml, 20X concentrated buffered saline containing detergents
      • TMB Substrate (Kit Component No. JA1608-12ML): 1 bottle, 12 ml, ready-to-use
      • Stop Solution (Kit Component No. JA1616-12ML):: 1 bottle, 12 ml, 2.5 N sulfuric acid, ready-to-use
      • Plate Sealers (Kit Component No. JB155-EA):: to cover plates during incubations
      • Lysis Buffer (Kit Component No. 71009-10ML):: 1 bottle, 10 ml, Cytobuster™ Protein Extraction Reagent (Cat. No. 71009)
      Materials Required but not provided• Precision pipettors with disposable tips
      • Wash bottle or multichannel dispenser for washing
      • PBS
      • Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/540 nm or 450/595 nm
      Preparation• Serum and Plasma: Remove all flocculent material from serum and plasma samples by centrifugation in a tabletop microcentrifuge. A 1:10 dilution of serum and plasma in Assay Buffer is recommended. • Cell Lysates: Harvest cells and centrifuge at 1000-3000 rpm to pellet the cells. Wash the cell pellet with ice-cold PBS and centrifuge to pellet the cells. Add 500-1000 µl Lysis Buffer (approximately 1 ml per 1x107 cells) and incubate on ice for 30 min. Vortex and centrifuge the lysate at 14,000 x g in a pre-cooled microcentrifuge. Immediately transfer the supernatant to a fresh microcentrifuge tube and discard the pellet. Dilute the lysate at least 1:10 before determining the protein concentration using a BCA protein assay. Dilute cell lysates in Assay Buffer in the range of 1:10-1:1000 prior to assay. Tissue Extracts: Use cytosol or detergent-based extracts.
      Reagent preparation• 1X Wash Buffer: Mix 1 part Wash Buffer Concentrate to 19 parts distilled or deionized water. For example, add 25 ml Wash Buffer Concentrate to 475 ml deionized or distilled water to make 500 ml 1X Wash Buffer. • Hsp27 Standard: Reconstitute each vial of Hsp27 Standard with 250 µl deionized or distilled water to yield a stock concentration of 1000 ng/ml. Unused, reconstitued Hsp27 Standard can be stored at -20° for 6 months. Frozen standard will withstand two freeze-thaw cycles. Generate a standard curve as outlined in the following table:

      Table 1: Concentration Guidelines

      *Please note that the Hsp27 Standard concentration is 2-fold lower in the wells due to mixing equal volume of Hsp27 Standard and Hsp27 Detection Antibody.

      • 1X Hsp27 Detection Antibody: Thaw the Hsp27 Detection Antibody at room temperature. Dilute 1:500 in Assay Buffer; dilute only the amount needed for the assay; 50 µl is required for each standard dilution and sample. The Hsp27 Detection Antibody solution will withstand two freeze-thaw cycles. • 1X HRP Conjugate: Dilute the HRP Conjugate 1:500 with Assay Buffer; dilute only the amount needed for the assay; 100 µl is required for each standard dilution and sample. Prepare just prior to use.
      Detailed protocol1. Remove the required number of strips and place them in the frame. Return the unused strips to the foil pouch and reseal entire edge of zip-seal.
      2. Add 50 µl of each Hsp27 Standard and sample and 50 µl of 1X Hsp27 Detection Antibody to designated wells. Cover with a Plate Sealer and incubate at 37°C for 2 h.
      3. Wash the wells with 1X Wash Buffer by completely filling the wells using a squirt bottle, multi-channel pipette, manifold dispenser, or autotomated plate washer. Completely remove liquid by shaking the contents into the sink. Repeat for a total of 4 washes. After the last wash, remove any remaining liquid by aspirating and tapping the inverted plate on paper towels. Complete removal of the liquid at each wash step is essential for good assay performance.
      4. Add 100 µl 1X HRP Conjugate to each well, cover with a Plate Sealer, and incubate at room temperature for 1 h.
      5. Wash the wells 4 times as outlined in step 3 above.
      6. Add 100 µl TMB Substrate to each well and incubate at room temperature for 15-20 min. A blue color will develop.
      7. Add 100 µl Stop Solution to each well. The color will change from blue to yellow.
      8. Read the absorbance at 450 nm, with the correction wavelength set at 540 or 595 nm.
      CalculationsSeveral options are available for calculating the concentration of Hsp27 in the samples. We recommend the use of a software package that will generate a 4-parameter logistic (4-PL) curve-fit. If data reduction software is not available, construct a standard curve by plotting the mean absorbance of each standard dilution on the Y-axis and the standard concentration on the X-axis and draw a best-fit curve through the points on the graph. The data may be linearized by plotting the log of the Hsp27 concentration vs. the log of the absorbance and the best-fit determined by regression analysis. Important Note: to determine the amount of Hsp27 in the samples the concentration derived from the standard curve must be multiplied by a factor of 2 [to account for the dilution obtained by mixing equal volumes of standard/sample and 1X Hsp27 Detection Antibody] and then by the initial sample dilution factor.
      Standard curve

      Figure 1: Example Standard Curve

      Hsp27 Standard dilutions were prepared as outlined in the Reagent Preparation section above and subsequently assayed according to the Detailed Protocol. The concentrations shown on the X-axis represent the concentrations in the wells as indicated in Table 1.
      Example data

      Table 2: Example Data

      Following the Detailed Protocol as outlined above, the concentration of Hsp27 was measured in 8 human cell lines, human breast tissue (normal and tumor), plasma, and serum.
      Assay Range0.02-1 ng/ml
      Recovery

      Table 3: Recovery

      Plasma, serum, and cell lysates were spiked with recombinant Hsp27 and subsequently assayed as outlined in the Detailed Protocol. The percent recovery of the spiked samples is shown in the table above.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ and Cytobuster™ are trademarks of EMD Chemicals, Inc.