anybody an expert on Fiske & Subarrow assay?

Engelbert Buxbaum EBUXBAUM at man.ac.uk
Wed Oct 18 06:44:14 EST 1995


In article <jnakamot-1410952319540001 at ts37-10.wla.ts.ucla.edu>, 
jnakamot at ucla.edu says...
>
>I have a friend who is trying to calibrate his Westerns 
(normalize for
>differing amts of sarcolemma in fetal vs. adult cardiac 
membranes) by
>measuring 
>Na+-K+ ATPase activity in the membrane extracts. He's basically 
trying to
>measure ouabain-sensitive Pi release using the Fiske & Subbarow 
assay for
>inorganic phosphate, but hasn't been having much luck, even 
using RBC
>membrane preps on which to troubleshoot and get the assay 
working. 
>
>Anybody out there who is both familiar with this or an 
equivalent
>phosphate assay (or, even better, who has experience measuring 
Na+-K+
>ATPase activity in cardiac membranes) and is willing to field a 
few
>questions from my friend? Or perhaps supply a detailed protocol 
of what
>works for you? (Yes, he has the pertinent Meth Enzymol and even 
the
>original articles). 
>
>Thanks,
>Jon
>
>Jon Nakamoto, M.D.
>Pediatrics/Endocrinology
>UCLA Medical Center
>jnakamot at pediatrics.medsch.ucla.edu 
>jnakamot at ucla.edu
>fax 310-206-5843
>
>-- 
>Jon Nakamoto
>jnakamot at ucla.edu
I used to do enzyme kinetics with the sodium pump with a coupled 
spectrophotometric assay (faster and simpler than phosphate 
determination): Schoner et al., Eur.J.Biochem. 1 (1967) 334. 
Alternatively, when the concentration of sodium pump in the 
sample is very low (like tissue homogenates), it may be better 
to use a fluorescent assay (hydrolysis of methylfluorescein 
phosphate): Noorgard et al., Biochim.Biophys.Acta 770 (1984) 203




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