05x_QUANT OF PROTEIN_BRADFORD_111309

05x_QUANT OF PROTEIN_BRADFORD_111309 - Calculation of...

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Calculation of Protein Concentration LABORATORY EXERCISE #3 Rev: 07:14:31 Calculation of Protein Concentration It is necessary to know the concentration of protein in order to use it for immunization and in assays. There are many colorimetric procedures for determining protein concentration, the Bradford method provides a convenient, rapid, and sensitive means of protein quantitation using the protein-binding dye Coomassie Blue WHY?. This dye undergoes a color change from brown to blue after binding to protein ( figure). The amount of blue color can be determined spectrophotometrically by absorbance at a wavelength of 595 η m. If the dye is added to a set of protein standards of known concentration, a standard curve can be constructed by plotting the absorbance reading (optical density) versus protein concentration. The concentration of protein in a sample can then be derived by measuring its absorbance and extrapolating the concentration from the standard curve. It is important that the absorbance reading for the unknown sample (the Ag preparation) fall within the range of the standard curve, and below a reading of 1.0 O. D. If a sample is too concentrated, several dilutions of the original sample will need to be prepared to find one that falls within the curve. Materials : - 100 mL Bio-Rad protein assay reagent (containing Coomassie Blue) - diluted 1:5, i.e. 20 mL of concentrated dye added to 80 mL of dH 2 O - 1 mL standard protein solution: bovine serum albumin (BSA, Sigma #A6793), 1.0 mg/ml in saline - 25 mL 0.9% (wt/vol) saline solution - 12x75-mm disposable test tubes (about 20) - Pipette gun and tips - Spectrophotometer (visible range) NOTE : All glassware should be thoroughly rinsed in distilled water before use, as many laboratory detergents react with Bradford solution Procedure : Bradford Assay - The “blank” : - Prepare one 12x75-mm tube containing 100 μ L of saline - BSA standard curve : Prepare six two-fold dilutions (i.e. 0.005 to 0.03 mg) of the standard protein BSA in saline: 1. Prepare six 12x75-mm tubes containing: 95, 90, 85, 80, 75, and 70 μ L of saline 2. Respectively, add: 5, 10, 15, 20, 25, and 30 μ L of 1mg/mL of BSA stock solution - Total volume in each tube should be 100 μ L 1
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- Experimental Samples – WHOLE CELLS : 1. Prepare four 12x75-mm tubes containing: 50, 25, 12.5, and 0
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This note was uploaded on 03/09/2010 for the course BIOL 371 taught by Professor Eugenemuller during the Fall '09 term at Framingham State College.

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05x_QUANT OF PROTEIN_BRADFORD_111309 - Calculation of...

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