The problem is that if you put detergent or reducing agent in Bradford or BCA, the reaction is complet also without protein. You can't determine the color gradient because every tubes are blue or purple.
________________________________________ De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Niks [nik...@gmail.com] Envoyé : vendredi 14 février 2014 07:45 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Determining concentration of membrane protein 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<mailto: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<mailto: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"
