[Protein-analysis] Re: (no subject)
Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Fri Nov 14 11:28:36 EST 2008
Am 09.11.2008, 04:26 Uhr, schrieb Vahid Chamanara
<vahidchamanara from gmail.com>:
> i want the true and simple methode to determaine and assay of ATPase
> activity for fish fillet.
The most sensitive determination was originally proposed by Neufeld & Levy
(1969) and uses gamma-33P (or 32P) labelled ATP. The radioactive phosphate
produced is, in the presence of carrier phosphate, converted into
phosphomolybdic acid and extracted into organic solvent. A modern (small
volume, non-toxic solvents) version of this assay I have described in Eur.
J. Biochem. 265 (1999) 54-63. With that assay you can detect fmol of
released phosphate in a few ul of sample.
If you can not work with radioactivity or if you can afford some loss of
sensitivity in exchange for much lower costs, you can use a colorimetric
assay. The phosphate released is converted to phosphomolybdic acid, which
is reduced to molybdenum blue (Fiske & Subbarov 1925). To increase the
sensitivity further you can bind dyes to the molybdenum blue (Ekman &
Jäger: Anal. Biochem. 214 (1993) 138; Kagami & Kamya: Meth Cell Biol 47
A continuous fluorescent assay for phosphate was described by Brune et al
in Biochemistry 33 (1994) 8262, but I have never used it.
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