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BC367L_2_2011

# BC367L_2_2011 - BC 367 Experiment 2 Comparison of Protein...

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BC 367 Experiment 2 Comparison of Protein Assays Introduction Proteins perform a range of complex functions in nature, including roles in structure, transport, and catalysis. When studying proteins, a biochemist often needs to determine protein concentration in a complex mixture of other molecules. Since there are numerous methods to measure protein content, it is critical to understand the advantages and disadvantages of each method available. In this exercise, you will use three different assays to determine the concentration of a standard protein. By sharing data for other proteins with other groups, you will compare the relative merits of each assay and establish how protein composition affects the sensitivity of a particular assay. Absorbance Spectroscopy All protein concentration determination methods depend upon measuring the absorbance of a solution. Absorbance (A) is a logarithmic measurement of the Transmittance (T) of light through a sample (Equation 1). Because Absorbance is a logarithmic function, values greater than 1.0 (10% T) or smaller than 0.1 (80%T) make it very difficult for the instrument to accurately determine the intensity of transmitted light. Solutions that do not absorb between 1.0 - 0.1 should be diluted or concentrated to give accurate data. A = - logT Equation 1 Chromophores in dilute solutions typically show a linear dependence between concentration and Absorbance (Equation 2). This functional dependence, known as the “Beer-Lambert law”, indicates that Absorbance is dependent upon the path length of light (l, in cm) through the sample, the concentration of the chromophore (c, in M), and the molar extinction coefficient ( ε , in M -1 cm -1 ). The path length and concentration can easily be changed, but the extinction coefficient is an intrinsic physical property of the chromophore. A = ε c l Equation 2 Because the relationship between the absorbance of the chromophore and its concentration is linear, it is possible to construct a calibration curve of absorbance versus concentration. An unknown concentration can then be determined from the absorbance of solution in question and the equation of the best-fit line. It is important to note that the unknown concentration should lie in the concentration range of the standard solutions for an accurate concentration determination. Protein Concentration Assays : Brief descriptions of a few of the more commonly used methods of protein determination and some of their limitations are given below. 1. BCA Assay: In alkaline solutions, Cu 2+ binds to peptide bonds of proteins. Cys , Trp , and Tyr , are capable of reducing the bound Cu 2+ to Cu + , resulting in formation of a moderate purple color proportional to the protein concentration. This color is used in the rather insensitive “Biuret Assay” to determine protein concentration. The sensitivity can be increased by addition of bicinchoninic acid (BCA). When BCA binds Cu + , an intense purple color proportional to the protein concentration is observed. However, because the composition of reducing amino acids

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