The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range. Equipment In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximum transmission in the region of 595 nm, on the border of the visible spectrum (no special lamp or filter usually needed).

The change in the color of Coomassie G-250 from red to blue upon binding protein is measured spectroscopically. In the absence of protein, when the dye is red, Bradford reagent has an absorbance maximum (A max) of 470 nm. In the presence of protein, the change to the anionic blue form of the dye shifts the A max to 595 nm.

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9 rows  · The method is based on the proportional binding of the dye Coomassie to proteins. …

22/05/2018  · Step 4: Measure absorbance, with spectrophotometer set at 595 nm . The data obtained here can be used to create a graph, with the absorbance on the y-axis and the known protein concentration on …

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max = 595 nm) (Reisner et al. 1975, Fazekes de St. Groth et al. 1963, Sedmack and Grossberg 1977). It is this blue protein-dye form that is detected at 595 nm in the assay using a spectrophotometer or microplate reader. H +H Cation « Neutral form « Anion 470 nm (red) 650 nm (green) 595 nm (blue) 2 4110065A.qxp 9/25/2007 2:39 PM Page 8

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