Lab 4.pdf - BIOL2070H Cell Biology Lab 4 PROTEIN...

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1 BIOL2070H Cell Biology Lab 4 PROTEIN QUANTIFICATION USING THE BRADFORD ASSAY Learning Objectives At the end of this lab, you will be introduced to the following skills and will learn how to: 1.Use pipettors to make dilutions 2.Use a spectrophotometer to measure protein concentrations 3.Generate a standard curve based on known concentrations of bovine serum albumin (BSA) 4.Use the standard curve generated above to determine the protein concentration in an unknown sample Introduction The Bradford Protein Assay, which measures the protein concentration of a solution, was developed by Dr. Marion M. Bradford in 1976.1The assay measures the binding of Coomassie Brilliant Blue G-250 dye to proteins. There are three forms of this dye, each with a different colour. Under acidic conditions, the dye exists in a red cationic form with a max absorption of 470 nm. When the dye binds to protein, it is converted to an unprotonated blue form with a max absorption of 595 nm. It is this form that is detected at 595 nm in a spectrophotometer. The key to the Bradford Protein Assay is the creation of a standard curve that displays the linear relationship between absorbance and concentration. This standard curve is generated by using a purified protein at a known concentration and measuring the absorbance of this solution at A595 nm using a spectrophotometer. The two most common standards used for protein assays are bovine serum albumin (BSA) and gamma-globulin. One of the drawbacks of the Bradford Protein Assay is that detergents (e.g., SDS) present in some cell

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