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Experiment+02+_+v+W12 - 2-1 Experiment 2 SPECTROPHOTOMETRIC...

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2-1 Experiment 2 SPECTROPHOTOMETRIC METHODS: PROTEIN DETERMINATION Introduction Chemical analyses are part of almost every investigation in biochemistry. The substances to be analyzed are present in milligram, microgram, or nanogram amounts, often below the detection limits of classical gravimetric and volumetric procedures. Since these compounds are usually in complex mixtures, assays must be selective, analyzing only the compound of interest. There are several spectrophotometric assays for determining protein concentrations. Each differs in sensitivity, specificity, and convenience. This experiment will introduce three commonly used methods: the absorbance of ultraviolet light (A280 method); the Bradford method; and the bicinchoninic acid (BCA) method. Objectives 1. Perform two spectrophotometric protein assays, prepare standard curves, and understand Beer's Law. 2. Compare spectrophotometric protein assays for sensitivity and potentially interfering substances. Theoretical Basis for Spectrophotometric Protein Assays Spectrophotometric assays are designed to determine the concentration of a substance using Beer’s Law. Often, the concentration of a substance is determined by comparing an experimentally derived absorbance of the substance (of unknown concentration) against the absorbance of a set of solutions with known concentrations of the substance (or a related substance), measured under the same assay conditions. The set of known concentrations of a substance is called the standards and its relationship to its measured absorbance is called the standard curve . Figure 2.1 are examples of standard curves. To construct a standard curve for protein concentration determinations, a series of protein solutions with a range of known concentrations is prepared, which are measured under identical conditions and the results are plotted as absorbance vs. concentration. Thus, the absorbance of a sample of unknown protein concentration is located on the ordinate of the standard curve (the y-axis) and its corresponding concentration is determined on the abscissa (the x-axis). The assays shown in Figure 2.1 either strictly obeys Beer's Law (right graph) where the relationship between absorbance and protein concentration is linear, or has a nonlinear relationship (left graph). This failure to obey Beer's Law perfectly can be of chemical or physical origin. However, a nonlinear graph is just as useful. By taking many points, the standard curve is more precisely defined. Regardless of the nonlinearity and linearity of a particular standard curve, either curve is rendered useless when (i) the absorbance exceeds the limits of the spectrophotometer (Abs 2.0), and (ii) when the absorbance exceeds the limits of the system ( ie when increased concentrations of protein no longer leads to significant increases in absorbance, the curve becomes flatter; or when lower concentrations of protein are below the level of detection and the absorbances do not change and, or are near zero).
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