SDH activity

• Purification of Mitochondria


  1. Implement the provided protocols to isolate mitochondria from cells

  2. Assess if the protocols effectively isolate functional mitochondria
• Data Analysis


  1. Analyze the provided data (titled 'Dataset X')

  2. Measure succinate dehydrogenase activity as a mitochondrial marker
• Specific Activity


  Perform calculations to determine the specific activity of SDH
• Presentation


  1. Present the standard curve graph in the report

  2. Include detailed calculations and values of specific activity
• Protocol Evaluation


  1. Assess if the used protocols are optimal for isolating mitochondria

  2. Propose improvements or alternative methods if necessary
• Protocol Overview


  1. Cell Lysis: Use mechanical, chemical, or enzymatic methods to break open cells

  2. Centrifugation: Perform differential centrifugation to separate mitochondria from other cellular components

  3. Purification: Further purify mitochondrial fractions using density gradient centrifugation
• SDH Activity Assay


  • Succinate + FAD → Fumarate + FADH2
  • Track the reduction of FAD to FADH2, which correlates with SDH activity
  • Use spectrophotometry to measure changes in absorbance
• Calculating Specific Activity


  1. Plot a standard curve of absorbance versus known concentrations of nmoles per well

  2. Remove background absorbance from all readings

  3. Obtain the equation for the line of best fit from the standard curve data points

  4. Calculate the change in absorbance over time for the sample

  5. Use the line of best fit equation to find the concentration (nmoles/well) in the sample

  6. Record the exact time the reaction was running

  7. Specific activity=nmoles/well in the sample/reaction time (min) * volume of sample in well
• Specific Activity


  Expressed as nmoles/min/mL (nanomoles per minute per milliliter)
• Critical Analysis


  1. Assess if the current method is the best for calculating specific activity

  2. Compare with other methods such as direct enzyme kinetics or alternative assays

  3. Consider the pros and cons of each method

  4. Evaluate if mitochondria were successfully isolated based on purity and activity

  5. High specific activity of SDH indicates successful isolation
• Subcellular Fractionation is a key technique for isolating cellular components, especially mitochondria
• SDH Activity Measurement is used as a marker for mitochondrial function
• Specific Activity is a critical parameter for assessing enzyme efficiency
• Data Analysis is essential for validating experimental protocols and results