Bradford Assay for Protein ● Introduction Spectrophotometry analysis of Proteins Spectrophotometry: is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through a sample solution. Used for qualitative and quantitative determination of biochemical compounds. Opening for blank reading Detector and preamplifier Microwell Plate carrier Measurement signal Background reading point Fiber quide Light Source Introduction Spectrophotometry analysis of Proteins Proteins absorb UV at 280, due to the presence of aromatic amino acids (Phenylalanine, Tyrosine, and Tryptophan). no, not all have aromatic COO H₂N-C-H CH₂ Phenylalanine Aromatic R groups COO H₂N-C-H CH₂ OH Tyrosine COO™ I H₂N-C-H CH₂ C-CH NH Tryptophan Introduction Bradford Assay for Protein (Coomassie protein assay) ● • The Bradford assay: is a quick and accurate spectroscopic analytical method for measuring the concentration of protein in a solution. ● • In an acidic environment, proteins bind to Coomassie dye. This results in a spectral shift from the reddish-brown form of the dye (absorbance maximum at 465 nm) to the blue form (absorbance maximum at 595 nm). A Absorbance Spectrum Brad Ford ● Unstable Cationic CBB Na س 200 میں منة Protein CH3 OLCH₂ CH3 Stable Anionic CBB OD 0.8 0.6 0.4 0.2 ON 79 0 400 S 500 600 700 Wavelength in nm 800 ·lamda max Const↑ app mole molecular 900 قياس أقل عمية موجودة Coomassie dye-protein complex can be measured at any wavelength between 575 nm and 620 nm with an approximate 10% decrease in the measured amount of color (absorbance) compared to that obtained at 595 nm. Introduction Bradford Assay for Protein(Coomassie protein assay) Development of color in Coomassie dye-based protein assays has been associated with the presence of certain basic amino acids (primarily arginine, Lysine, and histidine). HC–NH C-NH I CH₂ I *NH,CH–C−0 Histidine CH NH I C=NH I NH I CH₂ I CH₂ I CH₂ *NH;CH=c–0 Arginine CHINH, CH₂ I CH₂ NH,CH–C−0 Lysine Consendrakon increase اللون بينج The number of Coomassie dye molecules bound to each protein is approximately proportional to the number of positive charges found on the protein./n

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