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QIA95 PhosphoDetect™ EGFR ELISA Kit

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

Synonyms: Active Epidermal Growth factor Receptor ELISA Kit

Detection Methods: Colorimetric
QIA95
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.

      We apologize for the inconvenience, but we do not currently have an alternative product.





      Can be used to measure phosphorylated EGF receptor and for in vitro screening of EGF receptor kinase inhibitors.
      Catalogue NumberQIA95
      Brand Family Calbiochem®
      SynonymsActive Epidermal Growth factor Receptor ELISA Kit
      Materials Required but Not Delivered• Graduated pipettes for volumes to 10 ml
      • 5 to 1000 µl adjustable micropipettes with disposable tips
      • Multichannel micropipetting apparatus if available
      • Beakers, flasks, and cylinders necessary for preparation of reagents
      • Microwell strip reader capable of reading at 450 nm with 620 nm as optional reference wavelength
      • Glass-distilled or deionized water
      • Calculator with program to perform linear regression analysis if available
      References
      ReferencesGamou, S. and Shimizu, N. 1995. FEBS Lett, 357, 161.
      Gamou, S., et al. 1989. Exp. Cell Res. 183, 197.
      Glenny, J., et al. 1988. J. Immunol. Methods 109, 277.
      Hirai, M., et al. 1988. J. Cell Biol. 107, 791.
      Behzandian, M.A. and Shimizu, N. 1985. Cell Structure and Function 10, 219.
      Hunter, T. 1984. Nature 311, 414.
      Barnes, D. 1982. J. Cell Biol. 93, 1.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsPre-Coated 96-Well Plate, Anti-Phosphotyrosine/HRP-Conjugate, Active EGF Receptor Standard, Sample Diluent, Substrate Solution, Receptor Extraction Buffer, Stop Solution, Wash Buffer Concentrate, Plate Covers, and a user protocol.
      Applications
      Biological Information
      Assay range0.078 - 5.0 fmol/ml
      Assay time2.5 h
      Sample TypeHuman serum or biological samples or cell lysates
      Physicochemical Information
      Sensitivity0.078 fmol/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 36/37/38

      Irritating to eyes, respiratory system and skin.
      S PhraseS: 26-36-45

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsPre-Coated 96-Well Plate, Anti-Phosphotyrosine/HRP-Conjugate, Active EGF Receptor Standard, Sample Diluent, Substrate Solution, Receptor Extraction Buffer, Stop Solution, Wash Buffer Concentrate, Plate Covers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA95 0

      Documentation

      PhosphoDetect™ EGFR ELISA Kit Certificates of Analysis

      TitleLot Number
      QIA95

      References

      Reference overview
      Gamou, S. and Shimizu, N. 1995. FEBS Lett, 357, 161.
      Gamou, S., et al. 1989. Exp. Cell Res. 183, 197.
      Glenny, J., et al. 1988. J. Immunol. Methods 109, 277.
      Hirai, M., et al. 1988. J. Cell Biol. 107, 791.
      Behzandian, M.A. and Shimizu, N. 1985. Cell Structure and Function 10, 219.
      Hunter, T. 1984. Nature 311, 414.
      Barnes, D. 1982. J. Cell Biol. 93, 1.

      Brochure

      Title
      Protein Kinase Assay and Detection Kits Brochure
      User Protocol

      Revision07-July-2008 JSW
      SynonymsActive Epidermal Growth factor Receptor ELISA Kit
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at 4°C.
      BackgroundThe epidermal growth factor (EGF) receptor glycoprotein (170 kDa) consists of a 130 kDa protein and 40 kDa of oligosacccharide moieties. The EGF receptor molecule is made up of four domains: a glycosylated extracellular domain that is the binding site of EGF and TGFα, a transmembrane domain, a tyrosine kinase domain, and an autophosphorylation domain. The tyrosine kinase domain is an intracellular domain which is homologous to the oncogene v-erbB product. The binding of EGF to the receptor results in DNA replication and cell proliferation. Binding leads to rupturing of the cell membrane, phosphorylation of the EGF receptor and internalization, pH change, enzyme activation, actin filament reorganization, and induction of oncogenic proteins. Although the exact mechanism of EGF signal transduction is not known, EGF binding entails tyrosine autophosphorylation and the phosphorylation of downstream proteins. After ligand binding, receptor half-life is decreased, in one example from 20 h to 5 h. The Active EGF Receptor ELISA kit measures the quantity of phosphorylated EGF receptor in human serum, biological samples, and cell lysates. It is useful for analysis of receptor phosphorylation and for in vitro screening of specific inhibitors of EGF receptor kinase.
      Materials provided• 96-Well Plate (Kit Component No. JA6300-1EA): 1 plate, coated with monoclonal antibody to human EGF receptor
      • HRP-Conjugated (Kit Component No. JA6301-1VL): 1 vial
      • EGF-Receptor Standard (Kit Component No. JA6302-1VL): 1 vial, lyophilized, with preservative
      • Wash Buffer Concentrate (Kit Component No. JA6303-50ML): 1 bottle, 50 ml, 20X (contains PBS with 1% Tween®-20 Detergent)
      • Sample Diluent (Kit Component No. JA6304-11ML): 2 vials, 11 ml
      • Substrate Solution (Kit Component No. JA6305-12ML): 1 bottle, 12 ml, contains Tetramethylbenzidine
      • Receptor Extraction Buffer (Kit Component No. JA6306-11ML): 1 bottle, 11 ml
      • Stop Solution (Kit Component No. JA6307-12ML): 1 bottle, 12 ml, contains 1 N Sulfuric acid
      • Adhesive Plate Cover (Kit Component No. JA6308-1EA): 2 covers
      Materials Required but not provided• Graduated pipettes for volumes to 10 ml
      • 5 to 1000 µl adjustable micropipettes with disposable tips
      • Multichannel micropipetting apparatus if available
      • Beakers, flasks, and cylinders necessary for preparation of reagents
      • Microwell strip reader capable of reading at 450 nm with 620 nm as optional reference wavelength
      • Glass-distilled or deionized water
      • Calculator with program to perform linear regression analysis if available
      Precautions and recommendations• As conditions may vary from assay to assay, a standard curve must be established for every run. Bacterial, fungal, or cross-contamination of either samples or reagents or may cause erroneous results. While disposable pipette tips, flasks and glassware are preferred, reusable glassware must be thoroughly washed and rinsed before use.
      • Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty and fill wells as indicated for each wash cycle and do not allow wells to dry out at any time.
      • The limit of detection of EGF-R defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus three standard deviations) was determined to be less than 0.078 fmol/ml (mean of 10 independent assays).
      PreparationCell Lysate Preparation for Attached Cells: 1. Culture cells on a 90 mm dish to confluency (~107 cells). 2. Remove the supernatant without washing. Add 1 ml of Receptor Extraction Buffer and recover the cell solution from the dish with a cell scraper. Transfer the solution to a 1.5 ml microcentrifuge tube. 3. Spin at 4°C for 5 min at 10,000 x g and collect the supernatant. 4. Cell lysate supernatant may be diluted up to 50 times depending upon the cell line. Cell Lysate Preparation for Cells in Suspension: 1. Transfer cells to a microcentrifuge tube and spin at 300 x g. 2. Remove the supernatant (no washing). Add 1 ml of Receptor extraction buffer and suspend the cells by pipeting. 3. Spin at 4°C for 5 min at 10,000 x g and use the supernatant as sample. Samples should be prepared each time of assay. If samples must be stored, freeze at -80°C. Human serum and plasma can be assayed with no or 2-fold dilution.
      Reagent preparation• Wash Buffer Add distilled or de-ionized water to 50 ml for a final volume of 1,000 ml. Mix gently to avoid foaming and transfer to a clean wash bottle. Bacterial or fungal contamination of samples or reagents may cause false results. The pH of the final solution will be 7.4. Wash Buffer is stable for 30 days at 2 to 25°C. • HRP-Conjugate: Reconstitute with 11 ml of distilled water. Mix gently every 10 min avoiding foam formation. • EGF-Receptor Standard Reconstitute with 1 ml distilled water. Mix slowly for ~10 min. The concentration (fmol/ml) is shown on the vial label.
      Detailed protocol1. Mix all reagents gently to avoid foaming.
      2. Determine the number of microwell strips required to test the desired number of samples and the appropriate number of blanks and standards. Each sample, standard, blank, and optional control sample should be assayed in duplicate. Remove plate coated with mouse monoclonal antibody to human EGF-R from holder and store in sealed foil bag at 2 to 8°C with desiccant.
      3. Add 100 µl of Sample Diluent, in duplicate, to the standard wells leaving the first wells empty. Prepare standard dilutions by pipetting 200 µl of EGF-Receptor Standard, in duplicate, into wells A1 and A2. Transfer 100 µl from wells A1 and A2 to wells B1 and B2 respectively. Mix the contents of wells B1 and B2 and transfer 100 µl to wells C1 and C2 respectively. Do not scratch the inner surface of the microwells. Repeat step five times, creating two rows of EGF-R standard dilutions. Discard 100 µl of the contents from the last microwells (G1, G2) used.
      4. Add 100 µl of Sample Diluent, in duplicate, to the blank wells.
      5. Add 100 µl of each sample, in duplicate, to the designated wells.
      6. Cover with a Plate Cover and incubate at 37°C for 1 h on a rotator if available.
      7. Remove Plate Cover. Wash the microwell strips three times with ~300 µl Wash Buffer per well with thorough aspiration of microwell contents between washes. Do not scratch the surface of the microwells. After the last wash, tap microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwell strips immediately after washing or place upside down on wet absorbent paper for not longer than 15 min. Do not allow wells to dry.
      8. Add 100 µl of HRP-Conjugate to all wells.
      9. Cover with a Plate Cover and incubate or 1 h at 37°C.
      10. Remove Plate Cover and empty wells. Wash microwell strips 4 times. Proceed immediately to next step.
      11. Pipette 100 µl of TMB Substrate Solution to all wells including blanks.
      12. Incubate the microwell strips at room temperature for ~15 min on a rotator. Avoid direct exposure to intense light. The point at which the substrate reaction should be stopped may be determined by the ELISA reader being used. Many ELISA readers record absorbance only up to 2.0 Abs (Absorbance) Therefore the color development and the substrate reaction must be stopped before positive wells are no longer properly recordable.
      13. Stop the enzyme reaction by quickly pipetting 100 µl of Stop Solution into each well, including blanks. The Stop Solution must be spread quickly and uniformly throughout the microwells to completely inactivate the enzyme. Results should be read immediately after addition of the Stop solution or within one hour if the microwell strips are stored at 4°C in the dark.
      14. Read absorbance of each microwell on a spectrophotometer at 450 nm (620 nm serves as the reference wave length; 610 nm to 650 nm is acceptable). Blank the plate reader using the blank wells. Determine the Abs of the samples and the EGF-Receptor Standards. Note: incubation without shaking may result in lower Abs values.
      Calculations1. Calculate the average Abs values for each set of duplicate standards and samples. 2. Create a standard curve by plotting the mean Abs for each standard concentration on the ordinate against the EGF-receptor concentration on the abscissa. 3. Determine the concentration of circulating EGF-receptor for each sample by finding the mean absorbance value on the ordinate and extending a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding EGF-receptor concentration. Calculation of samples with an Abs exceeding the range of the standard curve may result in incorrect/low EGF-receptor levels. Such samples will require further dilution to accurately quantitate the EGF-receptor level. Values obtained should be within the expected range of known EGF-receptor controls. 4. A sample standard curve is shown below. A standard curve must be prepared for each group of microwell strips assayed.

      Figure 1: Standard Curve
      Sensitivity0.078 fmol/ml
      Assay Range0.078 - 5.0 fmol/ml
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
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      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.