Mark,
A little more information on the protein and need would be nice. Is
it a large peptide, a small protein, or a recombinant protein? Do
you want real quantitative results or semi quantitative (like BCA,
Bradford, or Lowry which can be off by 20% or more relative to the
[BSA])? Do you want it to be rapid assay? Is it to measure the
protein concentration of a "pure" sample or to follow a
purification? Do you want it to be non-invasive (like the A280
measurement) or how much protein are you willing to waste?
Some thoughts for a pure sample:
1) If you denature a sample, and label the lysines and N-terminus
with a fluorophore (e.g., fluoroscein), you can unambiguously measure
the absorption or fluouresence emission. But that a longish
procedure and you lose your protein. If you have Cys residues,
labeling those with DTNB or mercurichrome can allow you to do the
same thing. You just need to ensure all labeling sites are accessible.
2) Many people who recombinantly express a protein without Trp
residues have mutated a Phe or Tyr residue to Trp. Thus, the protein
has a built-in means to measure protein concentration by Trp
absorption or fluorescence.
3) If you have protein to waste or it survives being lyophilized,
dialyze it into ammonium acetate, lyophilize it, then weigh it on a
microbalance.
Measuring protein concentration is not an exact science if you want
it to be rapid and not waste lots of your precious protein sample.
Good luck,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [EMAIL PROTECTED]
****************************************************************
On Thu, Aug 21, 2008 at 12:54 PM, Mark Hilge <[EMAIL PROTECTED]> wrote:
Dear all,
I would be glad to hear what (simple) method I should use to
determine protein concentrations as accurately as possible.
Presently, I'm measuring absorption at 280nm with a nanodrop
device. I either have 0 or 1 tryptophan and no activity test.
Many thanks in advance!
Best regards,
Mark
Mark Hilge
Protein Biophysics
NCMLS 274
3rd floor M850.03.035
Geert Grooteplein 28
6525 GA Nijmegen
The Netherlands
http://www.mark-hilge.com
Phone: 0031 24 36 10 525
Het UMC St Radboud staat geregistreerd bij de Kamer van Koophandel
in het handelsregister onder nummer 41055629.
The Radboud University Nijmegen Medical Centre is listed in the
Commercial Register of the Chamber of Commerce under file number
41055629.
