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The assay is first performed with various known concentrations of a substance similar to that being measured. For example a standard curve for protein concentration is often created using known concentrations of bovine serum albumin. The assay procedure may measure absorbance, optical density, luminescence, fluorescence, radioactivity, or something else. An assay for protein is called the Bradford assay; it is a colorimetric assay. The reagent coomassie brilliant blue turns blue when it binds to arginine and aromatic amino acids present in protein. The intensity of the colour is best measured at 595 nm, which is the maximum absorbance (Amax) frequency of the blue dye, using a spectrophotometer. In this case the greater the absorbance, the higher the protein concentration.
This data is used to make the standard curve, plotting concentration on the X axis, and assay measurement on the Y axis. The same assay is then performed with samples of unknown concentration. To analyze the data, one locates the measurement on the Y-axis that corresponds to the assay measurement of the unknown substance and follows a line to intersect the standard curve. The corresponding value on the X-axis is the concentration of substance in the unknown sample.
(pdf format) Bradford Protein Assay. Bio-Rad Quick Starttm Bradford Protein Assay Instructional Manual. Retrieved on May 31, 2005.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Standard_curve". A list of authors is available in Wikipedia.|