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CBA107 Human Pro-ISG15 ELISA Kit

Human Pro-ISG15 ELISA Kit: SDB (Sicherheitsdatenblätter), Analysenzertifikate und Qualitätszertifikate, Dossiers, Broschüren und andere verfügbare Dokumente.
CBA107
  
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      Übersicht

      Replacement Information

      Key Spec Table

      Detection Methods
      Luminescence
      Description
      Overview

      This product has been discontinued.





      This kit is intended for the quantitative measurement of Pro-ISG15 in human cell lysates, tissue extracts, and plasma/sera samples.
      Catalogue NumberCBA107
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Precision pipettes with disposable tips
      • Wash bottle or multi-channel dispenser for washing
      • CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) or equivalent cell lysis buffer
      • Microplate luminometer
      References
      ReferencesBektas, N., et al. 2008. Breast Cancer Research 10, R58.
      Desai, S. D., et al. 2006. Cancer Res 66, 921.
      Lenschow, D.J., et al. 2006. PNAS 104, 1371.
      Ritchie, K.J. and Dong-Er, Z. 2004. Seminars Cell Dev. Biol. 15, 237.
      Product Information
      Detection methodLuminescence
      Form96 Tests
      Format96-Well Plate
      Kit containsAnti- Coated 96-Well Plate, Pro-ISG15 Standard, ISG15 Detector Antibody, HRP-Conjugate, 400X, Assay Diluent, Luminol, Peroxide Solution, ELISA 20X Plate Wash Concentrate, ELISA Stop Solution, Plate Sealers,and User Protocol.
      Applications
      Biological Information
      Assay range~5-200 ng/ml Pro-ISG15
      Physicochemical Information
      Sensitivity~2 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 22-25-35-36/37/38-43-62

      Harmful if swallowed.
      Toxic if swallowed.
      Causes severe burns.
      Irritating to eyes, respiratory system and skin.
      May cause sensitization by skin contact.
      Possible risk of impaired fertility.
      S PhraseS: 24/25-26-36/37-45-A09

      Avoid contact with skin and eyes.
      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing and gloves.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Intended useThe Calbiochem® Pro-ISG15 ELISA Kit is intended for the quantitative measurement of Pro-ISG15 in human cell lysates, tissue extracts, and plasma/sera samples.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsUpon arrival store the entire contents of the kit at -20°C. Following initial use, store the Luminol and Peroxide Solution at 4°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAnti- Coated 96-Well Plate, Pro-ISG15 Standard, ISG15 Detector Antibody, HRP-Conjugate, 400X, Assay Diluent, Luminol, Peroxide Solution, ELISA 20X Plate Wash Concentrate, ELISA Stop Solution, Plate Sealers,and User Protocol.
      Specifications
      Global Trade Item Number
      Bestellnummer GTIN
      CBA107 0

      Documentation

      Human Pro-ISG15 ELISA Kit Analysenzertifikate

      TitelChargennummer
      CBA107

      Literatur

      Übersicht
      Bektas, N., et al. 2008. Breast Cancer Research 10, R58.
      Desai, S. D., et al. 2006. Cancer Res 66, 921.
      Lenschow, D.J., et al. 2006. PNAS 104, 1371.
      Ritchie, K.J. and Dong-Er, Z. 2004. Seminars Cell Dev. Biol. 15, 237.
      Anwenderprotokoll

      Revision22-June-2009 RFH
      Form96 Tests
      Format96-Well Plate
      Detection methodLuminescence
      StorageUpon arrival store the entire contents of the kit at -20°C. Following initial use, store the Luminol and Peroxide Solution at 4°C.
      Intended useThe Calbiochem® Pro-ISG15 ELISA Kit is intended for the quantitative measurement of Pro-ISG15 in human cell lysates, tissue extracts, and plasma/sera samples.
      BackgroundISG15 (interferon-stimulated gene-15) is a 17 kDa protein that is activated by interferon as a primary response to viral or bacterial infections and to other cellular stress events. The ubiquitin-like ISG15 is conjugated to intracellular target proteins. Unlike ubiquitylation, ISGylation does not target proteins for degradation, but rather seems to play a regulatory, enhancing-cellular response to stress stimuli. ISG15 has not been identified in lower eukaryotes, indicating its role in specialized functions. Upon interferon treatment, ISG15 can be detected in both free and conjugated forms, and is secreted from monocytes and lymphocytes where it can function as a cytokine, Pro-ISG15 (165 amino acids), the inactive precursor of ISG15 (158 amino acids). The precursor is processed at the C-terminus by an ISG15-specific protease, USP18. The mature form of ISG15 contains the conserved C-terminal di-glycine residues that are critical for conjugation to intracellular target proteins. Recent evidenced showed that ISG15 and its protein conjugates are overexpressed in many tumors, and alteration of the ISG15 system confers drug resistance to tumor cells. ISG15 and its conjugates antagonize the activity of the ubiqutitin-proteasome system and, ultimately, may play an important role in cell growth and differentiation.
      Principles of the assayThe Calbiochem® Pro-ISG15 ELISA Kit is a complete kit for the quantitative determination of human Pro-ISG15 in cell lysates, cell extracts, and body fluids. The 96-well plate is coated with rabbit monoclonal anti-human Pro-ISG15 as the capture antibody. This antibody does not recognize the activated (cleaved) or conjugated forms of ISG15. Standards and samples are added to the wells and Pro-ISG15 present in the sample binds to the capture antibody. Pro-ISG15 is then detected with a goat polyclonal anti-human ISG15 antibody followed with anti-goat IgG HRP conjugate. A luminol/peroxide substrate solution is added to the wells and light is generated in proportion to the amount of Pro-ISG15. A microplate luminometer is used to measure the light signal and the level of Pro-ISG15 in each sample is calculated from the standard curve.
      Materials provided• Anti-Pro-ISG15 Coated 96-Well Plate (Kit Component No. JA9575-1EA): 1 white plate coated with rabbit anti-Pro-ISG15, supplied as twelve 8-well strips
      • Pro-ISG15 Standard (Kit Component No. JA9576-1UG): 2 vials, 1 µg each
      • ISG15 Detector Antibody (Kit Component No. JA9577-12ML): 1 bottle, 12 ml, ready-to-use
      • HRP-Conjugate, 400X (Kit Component No. JA9578-50UL): 1 vial, 50 µl, supplied as a 400X solution
      • Assay Diluent (Kit Component No. JA7644-50ML): 1 bottle, 50 ml
      • Luminol (Kit Component No. JA9572-6ML): 1 bottle, 6 ml
      • Peroxide Solution (Kit Component No. JA9573-6ML): 1 bottle, 6 ml
      • ELISA 20X Plate Wash Concentrate (Kit Component No. JA1617-100ML): 1 bottle, 100 ml
      • Plate Sealers: 2 each
      Materials Required but not provided• Precision pipettes with disposable tips
      • Wash bottle or multi-channel dispenser for washing
      • CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) or equivalent cell lysis buffer
      • Microplate luminometer
      Reagent preparationAllow all kit components to reach room temperature prior to assay run. Prepare reagents immediately prior to use. The following table provides reagent preparation instructions to obtain a volume of reagent sufficient for one 8-well strip.

      Table 1: Reagent Preparation

      Preparation of Standard: Label a set of tubes from 1 to 8 and prepare dilutions of the Pro-ISG15 Standard as outlined in the table below:

      Table 2: Preparation of Standard
      Detailed protocol1. Remove the desired number of strips from the Anti-Pro-ISG15-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 of each diluted Pro-ISG15 Standard and sample to designated wells. Cover the wells with a Plate Sealer and incubate for 90 min at room temperature.
      3. Aspirate or decant the contents of each well and wash the wells by completely filling each well with 1X ELISA Plate Wash and subsequently removing the contents by aspirating or decanting. Repeat for a total of four washes. After the last wash, remove any remaining 1X ELISA Plate Wash by inverting the plate and tapping on clean paper towels. So not allow the wells to dry completely.
      4. Add 100 µl Anti-ISG15 Detector Antibody to each well. Cover the wells with a Plate Sealer and incubate for 90 min at room temperature.
      5. Wash the wells as outlined in step 3 above.
      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 wells as outlined in step 3 above.
      8. Prepare Chemiluminescent HRP Substrate as outlined in Reagent Preparation; mix briefly. Add 100 µl Chemiluminescent HRP Substrate to each well. Incubate for 1-5 min at room temperature.
      9. Determine the RLU of each well using a 96-well luminometer.
      Assay characteristics and examples

      Figure 1: Typical Pro-ISG15 Standard Curve

      A sample of ISG15 and the Pro-ISG15 Standard were prepared as outlined above and assayed as outlined in the Detailed Protocol. A standard curve was generated by using the POLARstar Optima Luminometer from BMG LABTECH with automatic gain setting. Cross-reaction with ISG15 is less than 10%.


      Figure 2: Sample Values for Various Cell Lysates

      Cell pellets of depicted cell lines were resuspended with Cytobuster™ Protein Extraction Reagent (Cat. No. 71009). and incubated for 30 min on ice. Supernatants were assayed as outlined in the Detailed Protocol and the quantity of Pro-ISG15 was calculated from a standard curve.


      Figure 3: Sample Values for Plasma Samples

      Plasma from 3 different donors was assayed for Pro-ISG15 as outlined in the Detailed Protocol above. The quantity of Pro-ISG15 was calculated from a standard curve.
      Sensitivity~2 ng/ml
      Assay Range~5-200 ng/ml Pro-ISG15
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ and CytoBuster™ are trademarks of EMD Chemicals, Inc.