Dear colleagues,
Thanks a lot for the many constructive suggestions! Attchached is
another response I got directly.
Best regards,
Mark
______________________________________________________________________________
Hello Mark,
for low absorbing proteins, the Ehresmann method is a good choice. This
empiric formula converts absorption into protein concentration:
(A228.5-A234.5)/3.14 = [protein] in mg/mL
The method was developed to determine whole protein concentration in
plant
extracts that also contain nucleic acid. These absorb the same at the
two
wavelengths, so the difference is proportional to the protein
concentration provided there are no other absorbing agents (such as
thiols, old MOPS buffer etc.). 3.14 is an empiric conversion factor
unrelated to pi.
Since at these wavelengths the amide bonds absorb, one can get away with
very little protein. I have tried this in a nanodrop, but not
exhaustively. If the monochromator can not step with 0.5nm, four
wavelengths may be used as approximation.
Ref.:
Ehresmann B, Imbault P, Weil JH.
Spectrophotometric determination of protein concentration in cell
extracts
containing tRNA's and rRNA's.
Anal Biochem. 1973 Aug;54(2):454-63.
HTH, regards,
Markus
______________________________________________________________________________________
Mark Hilge
Protein Biophysics
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