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CBA057 Hsp90α ELISA Kit

Hsp90α ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: Heat Shock Protein 90α ELISA Kit

Detection Methods: Colorimetric
CBA057
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      Quantitative sandwich immunoassay for measuring human Hsp90α protein in cell lysates, tissue extracts and biological fluids. It uses an Hsp90α specific monoclonal antibody as the capture antibody. This kit is specific for Hsp90α and does not cross-react with Hsp90β.
      Catalogue NumberCBA057
      Brand Family Calbiochem®
      SynonymsHeat Shock Protein 90α ELISA Kit
      Materials Required but Not Delivered• Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably with a reference wavelength set at 540-600 nm.
      • Wash bottle or multi-channel dispenser for washing
      References
      ReferencesEustace, B.K., et al. 2004. Nat. Cell Biol. 6, 507.
      Goetz, M.P., et al. 2003. Ann. Oncol. 14, 1169.
      Young, J.C., et al. 2001. J. Cell Biol. 154, 267.
      Ritossa, F., 1962. Experienta 13, 571.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format 96-well plate
      Kit containsAnti-Hsp90α coated 96-well Plate, Hsp90α Standard, Hsp90α Detector Antibody, HRP Conjugate, Assay Diluent, ELISA 20X Plate Wash Concentrate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol.
      Positive controlHsp90α
      Applications
      Biological Information
      Assay range0.6-40 ng/ml
      Assay time4 h
      Sample TypeCell lysates, tissue extracts and biological fluids
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® Hsp90α ELISA Kit is intended for the quantitation of human Hsp90α protein in cell lysates, tissue extracts, and biological fluids. It does not cross-react with Hsp90β.
      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 store the entire contents of the kit at -20°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAnti-Hsp90α coated 96-well Plate, Hsp90α Standard, Hsp90α Detector Antibody, HRP Conjugate, Assay Diluent, ELISA 20X Plate Wash Concentrate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA057 0

      Documentation

      Hsp90α ELISA Kit Certificates of Analysis

      TitleLot Number
      CBA057

      References

      Reference overview
      Eustace, B.K., et al. 2004. Nat. Cell Biol. 6, 507.
      Goetz, M.P., et al. 2003. Ann. Oncol. 14, 1169.
      Young, J.C., et al. 2001. J. Cell Biol. 154, 267.
      Ritossa, F., 1962. Experienta 13, 571.
      User Protocol

      Revision08-December-2008 RFH
      SynonymsHeat Shock Protein 90α ELISA Kit
      Form96 Tests
      Format 96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at -20°C.
      Intended useThe Calbiochem® Hsp90α ELISA Kit is intended for the quantitation of human Hsp90α protein in cell lysates, tissue extracts, and biological fluids. It does not cross-react with Hsp90β.
      BackgroundThe heat shock response was first described in 1962 and the associated heat shock proteins (HSPs) are named for the fact that synthesis of these proteins is increased following heat shock, which is contrary to the reduced synthesis of most cellular proteins under the same conditions. In addition to heat, these proteins are also modulated by oxidative stress, inhibitors of energy metabolism, fever, and inflammation. Members of the Hsp90 family of proteins are (Hsp90α, Hsp90β, Grp94/gp96 in the endoplasmic reticulum, and the recently discovered Hsp75/TRAP1 in the mitochondrial matrix. Hsp90α is more inducible than Hsp90β, while constitutive levels of Hsp90β are two-fold higher than Hsp90α. Recent discoveries revealed that many of the key signaling molecules found deregulated in human cancers, including tyrosine kinases and steroid receptors, require the action of the Hsp90 proteins. Hsp90 is an important, emerging drug target for anti-cancer agents because it is specifically activated in tumor cells where it is responsible for controlling multiple oncoproteins that are critical to the proliferation and survival of the tumor. As a result, Hsp90-targeted drugs may potentially inhibit the growth of a wide range of cancer cells in both solid tumors and blood-based cancers. The Hsp90α isoform, but not the Hsp90β isoforms, is expressed extracellularly on fibrosarcoma and breast cancer cells where it interacts with the matrix metalloproteinase, MMP-2 and promotes its activation.
      Principles of the assayThe Hsp90α ELISA Kit is a standard sandwich immunoassay for the quantitation of human Hsp90α in cell lysates, tissue extracts, and biological fluids. The kit uses an Hsp90α-specific mouse monoclonal antibody, pre-coated on the wells of a 96-well plate, as the capture antibody. Standards and samples are incubated with the capture antibody and unbound material is washed out of the wells. A rabbit polyclonal detector antibody is added, followed by a goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP). TMB substrate is added and is cleaved by the HRP-conjugate to yield a blue colored product. Sensitivity is increased by the addition of a sulfuric acid stop solution, which yields a yellow color. The absorbance is measured at 450 nm, preferably with reference wavelength set at 540-600 nm. A standard curve is generated and the level of Hsp90α in each sample is determined by comparison to the standard curve.
      Materials provided• Anti-Hsp90α Coated 96-well Plate (Kit Component No. JA9194-1EA): 1 plate, 96-wells supplied as six 2x8-well strips, pre-coated with mouse anti-human Hsp90α antibody
      • Hsp90α Standard (Kit Component No. JA9196-100NG): 2 vials, 100 ng each lyophilized recombinant Hsp90α (Cat. No. 385901)
      • Hsp90α Detector Antibody (Kit Component No. JA9195-15UL): 1 vial, 15 µl
      • HRP Conjugate (Kit Component No. JA7922-100UL): 1 vial, 100 µl, supplied as a 200X solution
      • Assay Diluent (Kit Component No. JA7644-50ML): 1 bottle, 50 ml, ready-to-use
      • 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, preferably with a reference wavelength set at 540-600 nm.
      • Wash bottle or multi-channel dispenser for washing
      Reagent preparationAllow kit components and samples to reach room temperature prior to assay. The following provides reagent preparation instructions to obtain the volume required for one 2x8-well strip. • 1X Plate Wash: Prepare Plate Wash 1X by adding 3 ml ELISA 20X Plate Wash Concentrate to 57 ml deionized water. Mix well. • 1X Hsp90α Detector Antibody: Prepare Hsp90α Detector Antibody 1X by adding 2 µl Hsp90α Detector Antibody to 1998 µl Assay Diluent. Mix well. • 1X HRP Conjugate: Prepare 1X HRP-Conjugate by adding 10 µl HRP Conjugate to 1990 µl Assay Diluent. Mix well. • Standards: Reconstitute the lyophilized Hsp90α Standard with 500 µl water to yield a final stock concentration of 200 ng/ml. Unused standard may be dispensed into aliquots and stored at -20°C. A standard curve should be generated with each assay. Prepare diluted Hsp90α Standards according to the following table:

      Table 1: Diluted Hsp90α Standards
      Detailed protocol

      Table 2: Summary of Procedure


      1. Remove the desired number of strips from the Anti-Hsp90α-coated 96-Well Plate and place them in the frame. Return the unused strips to the foil pouch, reseal the entire edge with tape, and store at 4°C.
      2. Add 100 µl diluted standards and samples to designated wells. Cover the plate with a Plate Sealer and incubate for 2 h at 37°C.
      3. Discard the contents of the wells in the sink and wash by filling the wells completely with 1X ELISA Plate Wash. Shake the contents of the wells into the sink; repeat for a total of 3 washes. The complete removal of liquid at the end of each wash is essential for good assay performance. Following the last wash, remove the remaining liquid by aspirating the contents and tapping the inverted plate on paper towels.
      4. Add 100 µl 1X Hsp90α Detector Antibody to each well. Cover the plate with a Plate Sealer and incubate for 2 h at at room temperature.
      5. Wash the plate as indicated in step 3.
      6. Add 100 µl 1X HRP Conjugate to each well. Cover the plate with 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 15 min at room temperature.
      9. Add 100 µl ELISA Stop Solution to each well and read the absorbance at 450 nm, preferably with a reference wavelength set to 595 nm. Note: 540 nm or 550 nm can be used as alternative wavelengths to 595 nm. If dual wavelength reading option not available, read only at 450 nm.
      10. Calculate the Hsp90α concentration. Several options are available for the calculation of the Hsp90α concentration in 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 ng/ml.
      Assay characteristics and examples

      Figure 1: Typical Standard Curve using the Hsp90α Standard

      Serial dilutions of the Hsp90α Standard were prepared as indicated in the Preparation of Reagents section. Hsp90β, His•Tag®, Human, Recombinant (Cat. No. 385903) was used as a negative control. The concentration of the Hsp90 proteins was determined as outlined in the Detailed Protocol. Assay range: 0.6-40 ng/ml. Lower limit of detection is ~0.5 ng/ml. No significant reactivity with Hsp90β was observed.


      Figure 2: Immunoprecipitation and Quantitation of Hsp90α in Cell Lysates

      Total cell lysate from H460 (lung adenocarcinoma cells) was prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the manufacturer's recommended protocol and the protein concentration was determined using the BCA method. The concentration of Hsp90α was determined by ELISA to be 32 ng Hsp90α/mg total protein. Hsp90α was then immunoprecipitated using Anti-Hsp90α Mouse mAb (Cat. No. 386040) and detected by probing with Anti-Hsp90α Rabbit pAb (Cat. No. CA1016).
      Assay Range0.6-40 ng/ml
      Registered TrademarksCalbiochem® and His•Tag® are registered trademarks of EMD Chemicals, Inc.
      Interactive Pathways™ and CytoBuster™ are trademarks of EMD Chemicals, Inc.