Harold V. Taylor wrote:
>> Hi,
> I'm looking for a reliable method to assay membrane proteins. Does
> anybody know a method or reference which would help?
There is no single assay which will work in all cases. Three methods are
commonly used:
1) Bradford Dye Binding assay: The fastes method, can be read after
mixing sample and reagent. Will be interfered with by many detergents.
Moderately sensitive. Several similar methods have been published, which
use other dyes than Coomassie Blue, like eosin or erythrosin. As these
modifications offer no real advantage they are rarely used.
2) Peterson's modification of the Lowry et al. method: Tedious, requires
lengthy incubations. Interfered with by some detergents and reducing
substances. Not very sensitive.
3) The BCA method: Can be quite sensitive. Requires incubation at 65
degrees, so that 96 well plates can not be used. Serious interference by
reducing substances, however, non-reducing detergents have little
effect.
Non of these methods is "first principle", i.e., you need protein
standards for comparison. All of them give somewhat different colour
yield for different proteins, which has caused some heated debates in
the past about correction factors appropriate for this or that protein.
If you have access to a fluorimeter, you may want to look into
fluorescent assays (OPA, fluorescamine) for protein, I have no
experience with those, but they claim very high sensitivity (this may
reduce interference by dilution!).
The Warburg method (absorbance of the aromatic amino acids at 280 nm) is
obviously unsuitable for membrane proteins because of light scattering,
it is also not sensitive enough for most applications.
There is a volume in Meth. Enzymol. on protein purification (edited by
Deutscher), which has a usefull chapter on protein assays.
