The absorbance of a newly discovered protein at 595 nm is determined, normalized with the appropriate baseline spectrum. If the protein concentration is to be directly calculated from this absorbance, what additional information is needed?

Use the following information to answer questions: The Bradford assay is a common analytical technique that is used to measure the concentration of protein in a solution. It is both a spectroscopic and colorimetric procedure that is based on the absorbance shift of the Coomassie Brilliant Blue G-250 dye (Figure 1). The dye exists in the three forms: cationic (red), neutral (green), and anionic (blue). Under the acidic conditions used to perform the assay, the red form of the dye binds the protein and is converted to a stable blue anionic form, disrupting the native state of the protein and... Show more

Use the following information to answer questions:

The Bradford assay is a common analytical technique that is used to measure the concentration of protein in a solution. It is both a spectroscopic and colorimetric procedure that is based on the absorbance shift of the Coomassie Brilliant Blue G-250 dye (Figure 1). The dye exists in the three forms: cationic (red), neutral (green), and anionic (blue). Under the acidic conditions used to perform the assay, the red form of the dye binds the protein and is converted to a stable blue anionic form, disrupting the native state of the protein and exposing its hydrophobic pockets. The hydrophobic regions of the protein bind to the nonpolar region of the dye, and the amine groups of the protein are placed near the negatively charged substituents on the dye. When protein binds the dye, the complex is stabilized and exhibits an absorbance maximum at 595 nm that is proportional to the amount of bound dye.

Though the Bradford assay is less susceptible to interference by other chemicals that might be present in the protein solution, the presence of the organic anionic detergent sodium dodecyl sulfate (SDS) can disrupt the Bradford assay, working by two different modes at low and high concentrations to impact the absorbance readings at 595 nm.

 

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