Your basic choices for protein assays are:
1. Alkaline copper methods (e.g., Biuret and micro-biuret)
2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays)
3. Hydrophobic dye methods (e.g. Bradford)
4. UV methods (e.g., A280, A230, A210, etc.)
Method 1 is least sensitive to amino acid composition, but is also has
highest detection limits. Thiols interfere. Method 2 is very
idiosyncratic with amino acid composition, and also subject to
interference by thiols. Method 3 is not usable in detergent solutions.
Method 4 has many inteferences as most everything absorbs in the far UV
region.
If you have some special protein cofactors, metals, chromophores, etc.
these can be exploited for better measurements. For ecample
metalloproteins are easy to quantify by ICP-OES or TXRF if they are
reasonably pure.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote:
Dear CC4BBers,
I am trying to figure out what is the best way to determine the
protein concentration of my membrane protein. My purified membrane
protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of
DDM=0.0076%).
After reading the friendly manuals and searching online, I've learned
that detergents interferes with assays like Bradford but can't find
good descriptions of what works best. For now, I am trying to estimate
concentration from absorbance at 280nm and using molar extinction
coefficients based on aromatic amino acids, but again suspect
detergent interference. I would like to know what other folks working
on membrane proteins are doing.
Thanks very much.
Raji
--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University
