I want to isolate mitochondria, can I confirm it without going for electron microscope
Hello Arbind,
you could confirm your mitochondrial preparation by SDS-PAGE/Western-Blotting/Immunodetection.
You would require several antibodies for this experiment, for example:
for detecting cytosolic protein contamination: anti-alpha-tubulin,
for detecting nuclear protein contamination: anti-calnexin
Mitochondrial protein control: anti-VDAC
I hope this information is helpful to you.
With best regards, Christian
sir is it possible to confirm purity of mitochondria by using specific marker
enzyme, succinate dehydrogenase.
Mitochondrial citrate synthase could be used for the determination of mitochondrial purity.
What is the tissue? Probable contaminants are lysosomes, endoplasmic reticulum and a few peroxisomes; depending on the tissue. I would put a few drops on glass slides, let them dry briefly, fix briefly in 1% glutaraldehyde, rinse 3 times, then stain either for cytochrome oxidase, or acid phosphatase, or glucose 6-phosphate dehydrogenase, or catalase at pH 10.5. Comparing the results should give a rough idea of the purity.
Hello Arbind,
no electron microscope is needed.
WB: in addition to VDAC (outer membrane), you can also use subunit IV of cytochrome oxidase (COX-IV), as mitochondrial marker (inner membrane).
for enzyme activity: monoamino oxidase, adenylate kinase and glutamate dehydrogenase are mitochondrial outer membrane, intermembrane space and matrix markers, rescpectively.
Notice that if you need to rule out any cytosolic contamination, besides immunoblotting (a-or b-tubulin (or actin) are good marker for cytosol) you should check the absence of cytosolic marker enzyme activities. In this regard, be careful in your choice. Some of the widely used enzymes, see L-LDH (L-lactate dehydrogenase) and PK (pyruvate kinase), cannot be considered anymore markers since the occurrence of mitochondrial isoforms was reported.
Best,
Gianluca
Since mitochondria can be spinned down in the centrifuge, the main contaminants will be other granules sedimenting at roughly the same speed and sucrose solution. Cytosolic proteins will remain in de supernatant, I expect.
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