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E_+Experiment+02_v+F10 - 2-1 Experiment 2...

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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 measured are present in milligram, microgram, or even nanogram amounts, which is well below the detection limit of classical gravimetric and volumetric procedures. Since these compounds are usually in complex mixtures, assays must be selective, measuring only the compound of interest. There are several spectrophotometric assays for protein concentration that differ in sensitivity, specificity, and convenience. This experiment will introduce three commonly-used methods: the A 280 method, the Bradford method, and the bicinchoninic acid (BCA) method. Objectives 1. Perform three spectrophotometric protein assays, prepare standard curves, and understand Beer's Law. 2. Compare spectrophotometric protein assays for sensitivity and potentially interfering substances. 3. Learn to prepare a spectrophotometric protein assay protocol for the BCA protein assay. Theoretical Basis for Spectrophotometric Protein Assays Spectrophotometric assays are designed to determine the concentration of a substance using Beer’s Law: A = •c•l, where A is the absorbance = log(I ε 0 /I), is the molar extinction coefficient (M ε -1 cm -1 ), c is the molar concentration of the absorbing species, l is the length of the light path in cm through the absorbing solution, and I 0 and I are the intensities of light incident upon and emerging from the absorbing solution. 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.
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