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protein assay on membrane preps in HEPES

jac55 at cas.org jac55 at cas.org
Thu Dec 22 06:37:26 EST 1994


In article 2ru at nntp1.u.washington.edu, bperigo at u.washington.edu (Bob Perigo) writes:
>I just realized that the 50 mMolar HEPES in which we make our membrane
>preps is interfering with our modified Lowrey protein determinations.  How
>do you get around that problem? I found nothing in the literature after
>the 1975 Anal.Biochem 67, p198 notation that it and TRIS and some other
>buffers do interfer.  Hope you can help.


     Just about any reactive group (including -OH, -NH2, and -SH) interferes 
     with Lowry.   You can either TCA ppt. your samples and resuspend them
     in aq. NaOH or use a completely different assay.  One I used to use on
     samples rich in membranes (mitochondria) was a quantitative dye-binding
     assay (Schaffner, W. and Weissmann, C. (1973), Analyt. Biochem., 56,
     502 - 514).  Samples are TCA pptd. onto nitrocellulose filters, stained
     with Amido Black, destained, and the stain eluted and measured.  It 
     worked down to 1 microgram (volume doesn't matter because you're
     ppting.) and ought to be scalable to large numbers of samples with
     dot-blot manifolds.
     
     Alec Chambers
      
	Not waving, but drowning.
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