A280 vs. protein assay?

MESALV00 at ukcc.uky.edu MESALV00 at ukcc.uky.edu
Sat Sep 17 12:11:44 EST 1994

In article <tom-1509941445240001 at>
tom at radical.chm.jhu.edu (Tom Tullius) writes:
>We have encountered an unusual situation, and I was wondering if anyone
>else has seen this. We are studying the monomer-dimer equilibrium of a
>protein. Gel filtration chromatography of the (purified) protein sample
>give two bands (detected at 280 nm) that have roughly equal integrals.
>However, a Bio-Rad protein assay on each peak indicates that there is
>approximately 20 times more protein present in the leading (high MW) peak
>(presumably the dimer). Could dimerization somehow be "quenching" the
>absorbance at 280 nm? Ideas or experience would be appreciated.
>Tom Tullius
>Department of Chemistry, Johns Hopkins University
>tom at adical.chm.jhu.edu
Dear Tom,
Perhaps your low MW peak contains a bound ligand with absorbance at 280 nm.
The band width of some UV detectors is quite wide, wide enough to pick up
nucleotides and other "non-280 absorbing compounds.  Try determining the
spectra of your peak.  Good luck.
Mike Salvucci
USDA-ARS, Lexington KY

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