No, because Bradford is based on the increase in absorbance when the dye moves from a hydrophilic environment to a hydrophobic one (like the protein interior, or like the interior of a micelle). When detergents are present in excess of their CMC, the change in absorbance from partitioning into the micelles is generally large compared to any signal due to protein binding; plus preparing a perfectly matched blank solution is challenging when dealing with protein-detergent solutions.
I second Michael's recommendation--BCA works well. On 14 Feb 2014, at 1:45 AM, Niks wrote: > Dear All, > May be a stupid question. But if we take buffer with detergent as control > (Blank), would not the difference in ODs using any of the methods used e.g. > Bradford assay, gives protein concentration? > > Regards > Nishant > > > On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett <rrowl...@colgate.edu> wrote: > Your basic choices for protein assays are: > Alkaline copper methods (e.g., Biuret and micro-biuret) > alkaline copper + molybdate methods (e.g., Lowry, BCA assays) > Hydrophobic dye methods (e.g. Bradford) > 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 >> > > > > > -- > "The most difficult phase of life is not when No one understands you;It is > when you don't understand yourself"
