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Bradford_Assay_Protocol - Bradford Assay for Protein...

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Bradford Assay for Protein Content/1 KLE 1/7/09 Bradford Assay for Protein Content Pre-Lab Before you begin, read through the experimental steps and determine the purpose of this experiment. Fill in the blanks to the question: “How does __________________________________________________________ affect ___________________________________________________?” Now, answer the question with your hypothesis: “If _________________________________ then ___________________________________________________________.” Background and Theory In this laboratory exercise, you will investigate the relationship between fat content and protein content in milk. You will perform a colorimetric assay that uses a series of protein standards and a spectrophotometer to set up a standard curve which will allow the determination of protein concentration in any unknown solution. A spectrophotometer is an instrument that measures the amount of light absorbed by a solution. The spectrophotometer contains a prism that splits light into individual wavelengths. A particular wavelength is then shone through a sample solution. A detector on the far side of the sample determines how much light has been absorbed by the sample. The value given on the display is called the optical density (O.D). There are no units for O.D., which is also referred to as absorbance. Optical density can be thought of as the measurement of how "dark" a substance is (the "darker" the substance, the more light is absorbed and the higher the O.D. reading). Because proteins do not absorb light in the visible spectrum (380-750nm), spectrophotometric methods to measure proteins require the addition of reagents that react with or bind to specific amino acids or bonds in the protein. The result is a color change that can be measured at some wavelength of visible light. The color intensity is directly proportional to the concentration of protein in the solution and shows a linear relationship when graphed. To summarize: darker color = more light absorbed = higher O.D. value = more protein The colorimetric assay we will use to determine protein concentration is called the Bradford Assay (because Bradford developed it). This assay uses a dye called Coomassie Brilliant Blue G-250. Coomassie dye alone absorbs light at 465nm, but Coomassie dye bound to protein absorbs light at 595nm. You will mix protein solutions and Bradford reagent (which contains the Coomassie dye) together and then read the O.D. at 595nm on the spectrophotometer.
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Bradford Assay for Protein Content/2 KLE 1/7/09 The color response of the Bradford reagent is non-linear over a wide range of protein concentrations. Therefore, to use this assay to determine protein concentration in unknown solutions, you must first set up a standard curve of known protein concentrations. Once a graph of O.D. vs. protein concentration is plotted for a
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