Bradford Method
Determining the Concentration of an Unknown Protein Solution with the use of the Bradford Method
Paul Alcala, Andrea Basa, Melissa Caagbay, Frances Cayomba
Department of Chemistry, University of Santo Tomas, Manila, Philippines
Abstract
The Bradford method used to determine the protein content of a certain solution (Menguito, 2010) and involves the acidic Coomassie Brilliant Blue G-250 as a coloring reagent. [1] The dye is originally pinkish-brown in color when it is in its acidic state. When protein is bound to the dye the color turns blue. In this experiment a standard was used to start the experiment, specifically the BSA stock solution. This standard served as the “control” group of the experiment. Due to the given standard and by the use of the spectrophotometer to determine the absorbance, it is clearly seen that the concentration and the absorbance are directly proportional to one another. This therefore, served as a guide in order to find out if the unknown sample being tested follows the given standard. A standard curve plot is also drawn, plotting A595 versus the conc. of BSA to be able to tell the unknown concentration of the sample. [1] In conclusion, the unknown protein solutions’ concentration and absorbance were both directly proportional to one another.
Introduction
Proteins are polymers of amino acids, they are also called polypeptides. These proteins contain building blocks, called amino acids. There are 20 amino acids that serve as the building blocks. [2] These amino acids contain many amino acid residues (remaining portions of amino acids) which are linked together by amide bonds called, peptide bonds. “Proteins provide structural framework for the cells, regulation of cellular function, and enzyme activity.” (Scheppler, 2000). [3] There are three methods in order to determine the protein concentration of a certain mixture. These are the Lowry Method, The Bradford Method, and the BCA Method. [3] Each of these
Paul Alcala, Andrea Basa, Melissa Caagbay, Frances Cayomba
Department of Chemistry, University of Santo Tomas, Manila, Philippines
Abstract
The Bradford method used to determine the protein content of a certain solution (Menguito, 2010) and involves the acidic Coomassie Brilliant Blue G-250 as a coloring reagent. [1] The dye is originally pinkish-brown in color when it is in its acidic state. When protein is bound to the dye the color turns blue. In this experiment a standard was used to start the experiment, specifically the BSA stock solution. This standard served as the “control” group of the experiment. Due to the given standard and by the use of the spectrophotometer to determine the absorbance, it is clearly seen that the concentration and the absorbance are directly proportional to one another. This therefore, served as a guide in order to find out if the unknown sample being tested follows the given standard. A standard curve plot is also drawn, plotting A595 versus the conc. of BSA to be able to tell the unknown concentration of the sample. [1] In conclusion, the unknown protein solutions’ concentration and absorbance were both directly proportional to one another.
Introduction
Proteins are polymers of amino acids, they are also called polypeptides. These proteins contain building blocks, called amino acids. There are 20 amino acids that serve as the building blocks. [2] These amino acids contain many amino acid residues (remaining portions of amino acids) which are linked together by amide bonds called, peptide bonds. “Proteins provide structural framework for the cells, regulation of cellular function, and enzyme activity.” (Scheppler, 2000). [3] There are three methods in order to determine the protein concentration of a certain mixture. These are the Lowry Method, The Bradford Method, and the BCA Method. [3] Each of these
References: [1] Menguito, C.A. (2010). Basic Laboratory Expermients in Biochemistry. University of Santo Tomas. [2] Pratt, C.W. & Cornely, K. (2011). Essential Biochemistry. 2nd ed. Page 7. USA:John Wiley & Sons, Inc. [3] Scheppler, J.A., Cassin, P.E., & Gambier, R.M. (2000). Biotechnology Explorations: Applying the Fundamentals. Page 157-158. Washington, DC: ASM Press. [4] Bradford Method. (2012). Retrieved July 14, 2012 from, http://www.scribd.com/doc/51367139/Protein-Assay-Using-Bradford-Method [5] BSA standards (2012). Retrieved July 16, 2012 from, http://www.piercenet.com/browse.cfm?fldID=02020108