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Description
Overview
Assay kit provides a unique combination of reagents useful for the isolation of a highly enriched mitochondrial fraction from the cytosol. Translocation of cytochrome c from the mitochondrial fraction to the cytosol is monitored by immunoblotting with the cytochrome c antibody provided with the kit.
Catalogue Number
QIA87
Brand Family
Calbiochem®
Materials Required but Not Delivered
• DMSO • PBS
References
Product Information
Form
100 Tests
Format
Immunoblot
Kit contains
Mitochondria Extraction Buffer, Cytosol Extraction Buffer, Protease Inhibitor Cocktail, Reducing Reagent, Cytochrome c Antibody, and a user protocol.
Contact with acids liberates very toxic gas. Toxic by inhalation, in contact with skin and if swallowed. Causes burns. Irritating to eyes, respiratory system and skin. Risk of serious damage to eyes.
S Phrase
S: 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 use
The Cytochrome c Release Apoptosis Assay Kit provides a unique combination of reagents useful for the isolation of a highly enriched mitochondrial fraction from the cytosol that can be used to detect cytochrome c by 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 Conditions
Upon arrival store the entire contents of the kit at -20°C. Once opened, store the Mitochondria Extraction Buffer and Cytosol Extraction Buffer at 4°C and the remaining components at -20°C.
Avoid freeze/thaw
Avoid freeze/thaw
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
Mitochondria Extraction Buffer, Cytosol Extraction Buffer, Protease Inhibitor Cocktail, Reducing Reagent, Cytochrome c Antibody, and a user protocol.
Cytochrome c Release Apoptosis Assay Kit Certificates of Analysis
Title
Lot Number
QIA87
User Protocol
Revision
23-May-2011 JSW
Form
100 Tests
Format
Immunoblot
Storage
Upon arrival store the entire contents of the kit at -20°C. Once opened, store the Mitochondria Extraction Buffer and Cytosol Extraction Buffer at 4°C and the remaining components at -20°C.
Intended use
The Cytochrome c Release Apoptosis Assay Kit provides a unique combination of reagents useful for the isolation of a highly enriched mitochondrial fraction from the cytosol that can be used to detect cytochrome c by Western blotting.
Background
Cytochrome c plays an important role in apoptosis. The protein is located in the space between the inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome c from the mitochondria into cytosol where it binds to Apaf-1. The cytochrome c/Apaf-1 complex activates caspase-9, which then activates caspase-3 and other downstream caspases. The Cytochrome c Releasing Apoptosis Assay Kit provides an effective means for detecting cytochrome c translocation from mitochondria into cytosol during apoptosis. The kit provides unique formulations of reagents to isolate a highly enriched mitochondria fraction from cytosol. The procedure is simple and easy to perform, no ultracentrifuging is required and no toxic chemicals are involved. Cytochrome c releasing from mitochondria into cytosol is then determined by Western Blotting using cytochrome c antibody provided in the kit.
Principles of the assay
The Cytochrome c Release Apoptosis Assay Kit provides a unique combination of reagents useful for the isolation of a highly enriched mitochondrial fraction from the cytosol. Cells are subjected to an apoptotic stimulus (or left unstimulated) and extracted using the provided extraction buffers. The translocation of cytochrome c from the mitochondrial fraction to the cytosol is then monitored by immunoblotting with the cytochrome c antibody provided with the kit.
Materials provided
• Mitochondria Extraction Buffer (Kit Component No. JA5200-10ML): 1 bottle, 10 ml • 5X Cytosol Extraction Buffer (Kit Component No. JA5201-20ML): 1 bottle, 20 ml • DTT (1 M) (Kit Component No. JA5203-110UL): 1 vial, 110 µl (Blue cap) • 500X Protease Inhibitor Cocktail (lyophilized)* (Kit Component No. JA5202-1EA): 1 vial (Red cap), add 250 µl DMSO, and mix well before use • Cytochrome c Antibody** (Kit Component No. JA5204-0.5ML): 1 vial, 500 µl (Green cap), 0.2 mg/ml, mouse monoclonal IgG2b that recognizes denatured human, mouse, and rat cytochrome c
Materials Required but not provided
• DMSO • PBS
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
• 1X Cytosol Extraction Buffer: Prepare 1X Cytosol Extraction Buffer by adding the 20 ml 5X Cytosol Extraction Buffer to 80 ml diH2O; mix well.
• Mitochondria Extraction Buffer Mix: Just prior to performing the assay, prepare enough Mitochondria Extraction Buffer Mix for the number of samples to be assayed; each sample requires 0.1 ml of the mix. To prepare 1 ml Mitochondria Extraction Buffer Mix, add 2 µl 500X Protease Inhibitor Cocktail and 1 µl DTT to 1 ml Mitochondria Extraction Buffer; mix well.
• Cytosol Extraction Buffer Mix: Just prior to performing the assay, prepare enough Cytosol Buffer Mix for the number of samples to be assayed; each sample requires 1 ml of the mix. To prepare 1 ml Cytosol Extraction Buffer Mix, add 2 µl 500X Protease Inhibitor Cocktail and 1 µl DTT to 1 ml Cytosol Extraction Buffer; mix well.
Detailed protocol
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. Collect cells (5 x 107) by centrifugation at 600 x g for 5 min at 4°C. 3. Wash cells with 10 ml ice-cold PBS. Centrifuge at 600 x g for 5 min at 4°C. Remove supernatant. 4. Resuspend cells with 1 ml Cytosol Extraction Buffer Mix. 5. Incubate on ice for 10 min. 6. Homogenize cells in an ice-cold tissue grinder. Perform the task with the grinder on ice. 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 of the 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 step 7. Otherwise, perform 10-20 additional passes using the tissue grinder. Excessive homogenization should also be avoided, as it can cause damage to the mitochondrial membrane, which triggers release of mitochondrial components. 7. Transfer the homogenate to a 1.5 ml microcentrifuge tube and centrifuge at 700 x g for 10 min at 4°C. 8. Transfer supernatant to a fresh 1.5 ml tube and centrifuge at 10,000 x g for 30 min at 4°C. 9. Collect supernatant as Cytosolic Fraction. 10. Resuspend the pellet in 0.1 ml Mitochondrial Extraction Buffer Mix, vortex for 10 s, and save as Mitochondrial Fraction. 11. Load 10 µg of each cytosolic and mitochondrial fraction isolated from each cell sample (i.e., induced cells and uninduced cells) on a 12% SDS-PAGE. Then proceed with standard immunoblot procedure and probe with Cytochrome c antibody (1 µg/ml is the recommended starting concentration).
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
