Storage

Store at -20°C. This kit is valid for one year from the date of purchase.

Notes

• Perform all steps at low temperatures.
• Small-scale preparations are more convenient and faster than large-scale ones, making it easier to obtain intact mitochondria.
• Disrupting cells without damaging subcellular organelles is the most crucial step in mitochondrial preparation. Compared to tissue blocks, cultured cells, especially adherent cells, are more difficult to lyse with a glass homogenizer. Therefore, use a small-capacity glass homogenizer and a well-fitting pestle to grind adherent cells. Under a light microscope, about 50% of cells should remain unbroken. Excessive grinding can damage the mitochondria, while insufficient grinding will lower the yield.
• Calculate the correct centrifugation speed based on the centrifugal force in g. The centrifugation speed can be precisely calculated for different centrifuges. The relative centrifugal force (RCF) value (in g) depends on the rotor speed (rpm) and the radius of rotation (r, in mm) and can be expressed using the formula: RCF = 1.11 × 10⁻⁶ × r × (rpm)².
• For Western Blot and 2D electrophoresis, mitochondrial lysates can be directly added to sample buffer.
• PMSF must be added within 2-3 minutes before adding the lysis buffer to the sample to prevent PMSF from becoming unstable and ineffective in aqueous solution.
• This kit is suitable for fresh tissue samples only and is less effective for previously frozen tissues.

Only for research and not intended for treatment of humans or animals

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