Hi Everyone, Thanks very much for your helpful responses and suggestions. I will use the BCA assay.
Cheers, Raji On Thu, Feb 13, 2014 at 11:27 AM, R. M. Garavito <rmgarav...@gmail.com>wrote: > Roger, > > While I agree with your list, the BCA assay does not use molybdate (as we > make it from scratch with bicinchoninic acid, sodium carbonate, sodium > bicarbonate, sodium tartrate, and cupric sulfate pentahydrate). For > membrane proteins, I prefer the BCA assay until the protein is pure enough > to use A280. > > Cheers, > > Michael > > ****************************************************************** > *R. Michael Garavito, Ph.D.* > *Professor of Biochemistry & Molecular Biology* > *603 Wilson Rd., Rm. 513* > *Michigan State University * > *East Lansing, MI 48824-1319* > *Office:* *(517) 355-9724 <%28517%29%20355-9724> Lab: (517) > 353-9125 <%28517%29%20353-9125>* > *FAX: (517) 353-9334 <%28517%29%20353-9334> > Email: rmgarav...@gmail.com <garav...@gmail.com>* > ****************************************************************** > > > > > On Feb 13, 2014, at 10:39 AM, 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 > > > > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
