Dear colleagues! We plan to isolate mitochondria from freshwater amphipods, but didn't find any methods in literature - the closest found was the method of isolation from whiteleg shrimp Litopenaeus vannamei.
The problem is - the amphipods are quite small - around 1 cm long, so it's hard to isolate the gut before mitochondria isolation.
Will it work if we use just the sample of 10 g (or is that too much?) of amphipods and blender to homogenize it in isolation medium? Or it is crucial to select only some parts - for example only the amphipods legs and antennas?
P.S.: we do not have chitinase, nor the chance to get it in time.
Most techniques to isolate mitochondria use differential centrifugation, which entails two steps of low-speed centrifugation to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts, followed by a degree of high-speed centrifugation to concentrate mitochondria and separate them from other organelles.
Most methods to isolate mitochondria rely on differential centrifugation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts followed by high speed centrifugation to concentrate mitochondria and separate them from other organelles.
Mitochondria purification involves cell lysis followed by separation of the organelles from the rest of the cellular components. Here, we use detergent to rupture the cell membrane of mammalian cells followed by differential centrifugation to enrich the organelles.
Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.
Most methods to isolate mitochondria rely on differential centrifugation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts followed by high speed centrifugation to concentrate mitochondria and separate them from other organelles.
Mitochondria purification involves cell lysis followed by separation of the organelles from the rest of the cellular components. Here, we use detergent to rupture the cell membrane of mammalian cells followed by differential centrifugation to enrich the organelles.
Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.
Isolating mitochondria from freshwater amphipods involves several steps, including homogenizing the tissue, centrifuging the homogenate to pellet the mitochondria, and separating the mitochondria from other cellular components. Here is a general protocol for isolating mitochondria from freshwater amphipods:
It is important to note that the specific details of the protocol may vary depending on the specific goals of the experiment and the resources available. It is also important to follow good laboratory practices and to handle the samples and chemicals carefully to avoid contamination and to ensure the integrity of the results.
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