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Unformatted text preview: Determining Protein Concentration
Using the Standard Curve Biuret Reagent The copper ions in Biuret Reagent react with the
peptide bonds of serum proteins to form a purple color with an absorbance maximum at 540nm. This method can be used to determine the amount of soluble protein in a solution. We will use the spectrophotometer to measure the intensity of the color produced. The more protein present, the darker the solution Creating a Standard Curve A standard curve is created using the
absorbances of protein solutions of known concentrations The absorbance readings are used to construct a graph of absorbance as a function of protein concentration This graph can then be used to convert absorbance readings of experimental samples into protein amount/concentration. Preparing the Samples Start with a stock
solution of BSA protein. Add the equal amount
of water as protein solution to create a zero tube for the spectrophotometer. Make a serial dilution in the range of protein that you are working with. It may be necessary to make more than one standard curve. Always incubate for equal times and take continuous readings once you begin. Standard Curve First, set up all of the
tubes and gather required equipment Add biuret reagent and allow time to incubate. Take readings of all samples, then plot the data on a graph. An Example of a Bradford Curve ...
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- Spring '08