Hi Everyone, Thank you so much for your additional tips about various kits. My protein is tagged and when I cleave off the tag (a step that needs reducing agent), I will unfortunately have both detergent and reducing agent in my protein buffer.
Nicolas, thanks for your word of caution. I can't therefore use Pierce RAC BCA kit but it looks like I should be able to use the "660 nm" kit recommended by Ho (Thanks very much, Ho!). By extension, is it then the case that the BCA assay is not recommended when both detergent and reducing agent are present or is that just a peculiarity of the Pierce RAC BCA kit? Thanks very much to everyone who responded! Raji On Fri, Feb 14, 2014 at 9:49 AM, Patrick Loll <pat.l...@drexel.edu> wrote: > 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: >> >> 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 >> >> >> > > > -- > "The most difficult phase of life is not when No one understands you;It > is when you don't understand yourself" > > > > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
