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QIA88 ProteoExtract® Cytosol/Mitochondria Fractionation Kit

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QIA88
  
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      Übersicht

      Replacement Information
      Description
      OverviewThis kit provides a unique combination of reagents useful for the isolation of a highly enriched mitochondrial fraction from the cytosol. The enriched fractions can be used to study factors of interest using Western blotting, ELISA, or other assays.
      Note: 1 Test = reagents sufficient for processing 5 x 107 cells.
      Catalogue NumberQIA88
      Brand Family Calbiochem®
      Application Data
      Mitochondrial and cytosolic fractions were isolated from HEPG2 cells using QIA88 ProteoExtract® Cytosol/ Mitochondria Fractionation Kit. Mitochondrial pellets were resuspended in PBS. The protein concentration of each fraction was determined using the BCA assay. DTT was omitted from the Cytosol Extraction Buffer as it can interfere with concentration determination in the BCA assay. Equivalent amounts (10 µg) of HEPG2 homogenate (total protein), cytosolic, and mitochondrial fractions were analyzed by Western blot for proteins localized to different cellular regions as indicated. Representative results from 2 independent fractionations are shown.
      Materials Required but Not Delivered• Phosphate buffered saline (PBS)
      • Dounce tissue homogenizer
      • DMSO (for reconstitution of the Protease Inhibitor Cocktail)
      References
      Product Information
      Form100 Extractions
      FormatCell extraction
      Kit containsMitochondria Extraction Buffer, Cytosol Extraction Buffer, Protease Inhibitor Cocktail, Reducing Reagent, and a user protocol.
      Applications
      Biological Information
      Sample TypeCells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 23/24/25-32-34-36/37/38-41

      Toxic by inhalation, in contact with skin and if swallowed.
      Contact with acids liberates very toxic gas.
      Causes burns.
      Irritating to eyes, respiratory system and skin.
      Risk of serious damage to eyes.
      S PhraseS: 23-26-36/37/39-45

      Do not breathe fumes.
      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Intended useThe ProteoExtract® Cytosol/Mitochondria Fractionation Kit is designed for extraction of mammalian cells to yield enriched mitochondrial and cytosolic fractions for use in downstream analyses, such as Western blotting.
      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. Following initial use, store the buffers at 4°C and the DTT and Protease Inhibitor Cocktail at -20°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsMitochondria Extraction Buffer, Cytosol Extraction Buffer, Protease Inhibitor Cocktail, Reducing Reagent, and a user protocol.
      Specifications
      Global Trade Item Number
      Bestellnummer GTIN
      QIA88 0

      Documentation

      ProteoExtract® Cytosol/Mitochondria Fractionation Kit SDB

      Titel

      Sicherheitsdatenblatt (SDB) 

      ProteoExtract® Cytosol/Mitochondria Fractionation Kit Analysenzertifikate

      TitelChargennummer
      QIA88

      Broschüre

      Titel
      Caspases and other Apoptosis Related Tools Brochure

      Literaturstellen

      Titel
      Periyakaruppan, A., et al. 2009. Arch. Toxicol. 83, 595.
    • Saquib A. Lakhani, et al. (2006) Caspases 3 and 7: Key Mediators of Mitochondrial Events of Apoptosis. Science 311, 847-851.
    • Rupesh Chaturvedi, et al., 2004. Induction of Polyamine Oxidase 1 by Helicobacter pylori Causes Macrophage Apoptosis by Hydrogen Peroxide Release and Mitochondrial Membrane Depolarization. Journal of Biological Chemistry 279, 40161.
      		•
      Anwenderprotokoll

      Revision21-December-2010 RFH
      Form100 Extractions
      FormatCell extraction
      StorageUpon arrival store the entire contents of the kit at -20°C. Following initial use, store the buffers at 4°C and the DTT and Protease Inhibitor Cocktail at -20°C.
      Intended useThe ProteoExtract® Cytosol/Mitochondria Fractionation Kit is designed for extraction of mammalian cells to yield enriched mitochondrial and cytosolic fractions for use in downstream analyses, such as Western blotting.
      Principles of the assayThe ProteoExtract® Cytosol/Mitochondria Fractionation Kit provides a unique set of reagents for mammalian cell extraction that yields highly enriched mitochondrial cytosolic fractions. The enriched fractions can be used for dowstream analysis by Western blotting, ELISA, or other assays. Ideal for studying translocation of key molecules in apoptotis and signal transduction pathways. No ultracentrifugation is required and no toxic chemicals are involved.
      Materials provided• Mitochondria Extraction Buffer (Kit Component No. JA5300): 1 vial, 10 ml
      • 5X Cytosol Extraction Buffer (Kit Component No. JA5301): 1 bottle, 20 ml
      • DTT (1 M) (Kit Components No. JA5303): 1 vial, 110 µl
      • Protease Inhibitor Cocktail (Kit Components No. JA5302: 1 vial, 250 µl upon reconstitution
      Materials Required but not provided• Phosphate buffered saline (PBS)
      • Dounce tissue homogenizer
      • DMSO (for reconstitution of the Protease Inhibitor Cocktail)
      Precautions and recommendations• Read the entire protocol before beginning the procedure.
      • Be sure to keep all buffers on ice at all times during the experiment.
      Reagent preparation• Protease Inhibitor Cocktail: Reconstitute the lyophilized Protease Inhibitor Cocktail by adding 250 µl DMSO; mix well before use. Dispense into aliquots and store at -20°C. • 1X Cytosol Extraction Buffer Mix: Prepare 1X Cytosol Extraction Buffer by diluting the Cytosol Extraction Buffer 1:4 with ddH2O. Prepare only the amount needed for the assay (5 x 107 cells require 1 ml 1X Cytosol Extraction Buffer Mix). Just prior to use add 2 µl Protease Inhibitor Cocktail and 1 µl DTT to 1 ml 1X Cytosol Extraction Buffer. • Mitochondria Extraction Buffer Mix: Prepare only the amount needed for the assay (5 x 107 pelleted cells require 100 µl Mitochondria Extraction Buffer Mix). Just prior to use, add 2 µl Protease Inhibitor Cocktail and 1 µl DTT to 1 ml of Mitochondria Extraction Buffer.
      Detailed protocol1. Grow cells using appropriate culture medium. Induce apoptosis by desired method. Concurrently incubate a control culture without induction.
      2. Collect 5 x 107 cells by centrifugation at 600 x g for 5 min, 4°C.
      3. Wash the cells by resuspending the pellet with 10 ml ice-cold PBS. Centrifuge the cells at 600 x g for 5 min, 4°C. Remove the supernatant and discard.
      4. Resuspend the cells with 1 ml 1X Cytosol Extraction Buffer Mix and incubate on ice for 10 min.
      5. Homogenize the cells using an ice-cold dounce tissue homogenizer. Maintain on ice during the homogenization procedure. 30-50 passes with the grinder are recommended; however, efficient homogenization may depend on the cell type.
      Note: To check the efficiency of homogenization, pipette 2-3 µl homogenized suspension onto a coverslip and observe under a microscope. A shiny ring around the nuclei indicates that cells are still intact. If 70-80% of the nuclei do not have the shiny ring, proceed to the next step. If the cells are not sufficiently homogenized, perform 10-20 additional passes using the dounce tissue grinder. Excessive homogenization should also be avoided, as it can cause damage to the mitochondrial membrane, which triggers release of mitochondrial components.
      6. Transfer the homogenate to a 1.5 ml microcentrifuge tube and centrifuge at 700 x g for 10 min, 4°C.
      7. Transfer the supernatant to a clean 1.5 ml microcentrifuge tube and centrifuge at 10,000 x g for 30 min, 4°C.
      8. Transfer the supernatant to a clean tube. This is the Cytosolic Fraction.
      9. Resuspend the pellet, obtained in step 7, in 100 µl Mitochondria Extraction Buffer Mix and vortex for 10 s. This is the Mitochondrial Fraction. Note: If intact mitochondria are desired, resuspend the pellet, obtained in step 7, in 100 µl PBS (not provided). These are the intact mitochondria.
      10. The fractions are ready for use in downstream applications, such as Western blotting, ELISA, or other assay.
      Example data

      Figure 1: Detection of Cytosolic and Mitochondrial Proteins

      Mitochondrial and cytosolic fractions were isolated from HEPG2 cells using QIA88 ProteoExtract® Cytosol/ Mitochondria Fractionation Kit. Mitochondrial pellets were resuspended in PBS. The protein concentration of each fraction was determined using the BCA assay. DTT was omitted from the Cytosol Extraction Buffer as it can interfere with concentration determination in the BCA assay. Equivalent amounts (10 µg) of HEPG2 homogenate (total protein), cytosolic, and mitochondrial fractions were analyzed by Western blot for proteins localized to different cellular regions as indicated. Representative results from 2 independent fractionations are shown.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      ProteoExtract® is a registered trademark of Merck, KGaA, Darmstadt.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.