To biochemically analyse the cell content,
some methods are employed to separate the nuclei, cytosol and mitochondria.
After isolating the nuclei, cytosol and mitochondria with centrifugation methods, assays are carried out to identify the 3 cellular portions.
To identify cytosol, the enzymatic activity of LDH is detected.
To identify mitochondria, the enzymatic activity of 5'-nucleotidase is detected.
To identify nuclei, the DNA content is detected.
The question are,
1.) how to detect the LDH activity?
2.) how to detect the 5'-nucleotidase?
3.) Ethidium Bromide can be used to bind to the DNA, and the fluoresence degree can be used to identify the DNA portion. But how does EtBr bind to the DNA?

It might perhaps be easier to probe for the markers (eg lamin in nuclei, aldolase in cytoplasm) by Western blotting - this gives you an idea of purity, and cross-contamination across the fractions, while the rest can be used to study components of your interest.

I did use a kit from pierce to separa the nuclei and the cytosol and I did get a great result.

Which is the Pierce kit - can you give me a cat no.? I've tried the regular buffer/needle based method - which is okay, and the Qiagen Qproteome system - which is good. However, there seems to be a slight membrane carryover.

It is NE-PER Nuclear and Cytoplasmic Extraction Reagent
cat# 78833

Thanks!

Hi everyone..:)

anyone has ever used ne per nuclear and cytoplasmic extraction kit, together with sample grinding kit by amersham?

thank you....