Detection of "Invisible" Proteins???
bernard at elsie.nci.nih.gov
Wed Oct 26 00:22:11 EST 1994
In article <38jv76$bjo at jhunix1.hcf.jhu.edu>, ntheo at welchlink.welch.jhu.edu (NICHOLAS THEODORAKIS ) writes:
> In article <ming-2410942139320001 at sweetprotein.ahabs.wisc.edu>,
> Ding Ming <ming at ahabs.wisc.edu> wrote:
> >In article <1994Oct24.191425 at opal.tufts.edu>, ggordon at opal.tufts.edu wrote:
[Article trimmed for brevity]
> >> Our problem is this...this sequence contains NO AROMATIC AA's...That
> >> means it can't be detected by UV-light, and therefore can't have its
> >> concentration determined by spectrophotometry.
> >> This is very frustrating, since we will get large numbers of fractions
> >> and would like to know which ones contain the optimal ammount of protein.
> >You can certainly use 214 nm for peptide bond absorption, why do you cry
> >for aromatic-less?:)
> Maybe they can't if their FPLC doesn't use wavelengths that low. If only
> they bought a real HPLC...
> Nick Theodorakis
(Probably because the metal will mess up their protein)
Anyway, one suggestion is to perform quantitative/semi-quantitative ninhydrin
analysis on your fractions. If you are looking for a *really* quick result
then just spot your fractions on a TLC plate and treat them with a ninhydrin
spray. There are a few other good quick tests, such as TNBS (as long as
you have some free amino groups). A TLC guide will give you the protocols
for the "quick" tests and a peptide chemistry guide will give you the
protocol for the quantitative assay (which is a bit more involved).
All the very best,
Bernard ("purple fingers")
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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