Best method to get Cytoplasm extract? - without mitochondria or nuclear extracts (Aug/24/2008 )

Hello,

I've read that Digitonin buffer is used to extract only the cytoplasm. I don't have Digitonin, so what other detergents can be used to extract cytoplasm?

RIPA , NP40, Triton X-100 and CHAPS extract everything, I don't want to use them.

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Use a non-detergent hypotonic buffer to swell the cells (10-20 min on ice) then mechanically break open the cells with dounce homogeniser or a needle (can check under microscope). Low speed spin (3000g) will pellet the nuclei and mitochondria.

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Hi michelle. I know you from the other post. you have previously told me about Dounce Homogenizer. I don't have this homogenizer. but I have another one from IKA. it has a small shaft that can even go into microtube of 1.5 ml. is it ok?

have you also heard of Digitonin Buffer to extract cytoplasm components?

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Sure, your homogeniser would work.

Never tried detergent for cytosol fractions, so can't comment.

Good luck.

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you need a loose-fit homogeneizer (potter); an alternative detergent to digitonin is saponin, however there is no need of chemical lysis if you homogeneize carefully...

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you mean the nuclei and mitochondria content won't release into the supernatant? I don't want the mitochondria content interfere with my result!

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Michelle, what is the recipe of Hyptonic buffer that you mentioned here?

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to get all the mitochondria pelleted spin at 12,000 x g. The huge mitochondria can come down at lower gs.

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thanks,

I found a good protocol. it is on page 351-353 of the book Mitochondrial DNA by William C something

uses digitonin, sucrose and mannitol

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