Recitation2Slides_BIOCH756Fall2017.pdf

Intensity of the purple color or absorbance due to

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Intensity of the purple color or absorbance due to purple color complex at 545nm can measure the protein concentration Lowry method ! Reaction of copper ions with peptide bonds under alkaline conditions ! Oxidation of aromatic residues ! The reduction of Folin-Ciocalteu (F-C) reagent ( Phosphomolybdic and Phosphotungstic acids), which subsequently causes a color change of the solution into blue with an absorption in the range of 650 to 750 nm detectable with a spectrophotometer. Cu 2+ + protein > Cu 2+ -protein complex (under alkaline condition) Cu 2+ + (Tyr,Trp, AA’s) red > Cu + + (AA’s) ox Cu + + (F-C) ox > Cu2+ + (F-C) red
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8/31/2017 4 Bicinchoninic acid assay (BCA) ! BCA stock is highly alkaline ! Bicinchoninic acid ! Sodium carbonate ! Sodium bicarbonate ! Sodium tartrate ! Copper(II) sulfate pentahydrate ! Cu 1+ - BCA coordination complex absorbance at 562nm Protein + Cu 2+ ! Cu 1+ Bradford method ! Absorbance shift of the dye Coomassie Brilliant Blue G-250 ! Under acidic conditions red to blue change ! Absorbance at 595nm ! A method for linearization of the Bradford protein assay has been published in the following article : Ernst, O., Zor, T. Linearization of the Bradford Protein Assay. J. Vis. Exp. (38), e1918, doi:10.3791/1918 (2010). Here is a video about it: http://www.jove.com/video/1918/linearization-of-the-bradford- protein-assay Coomassie Blue + protein > blue complex Common Procedures for Colorimetric Methods 1. Generate range of concentrations for protein standard (usually BSA). 2. Add fixed volume of standard to fixed volume of dye solution. 3. Incubate for specific time and specific temperature. 4. Measure Absorbance at single wavelength specific to method (i.e. specific to dye). 5. Plot Absorbance vs. [BSA standard]assay in mg/mL (= standard curve). 6. Repeat process for the unknown protein.
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8/31/2017 5 Summary Colorimetry and spectrophotometry are very sensitive, simple, potentially non-destructive and highly informative analytical methods for quantitative analyses of biomolecules.
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