330 lab 2007 spring B part

330 lab 2007 spring B part - Spectrophotometric...

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    Objectives: To develop a familiarity with spectrophotometry and operations of spectrophotometer. - Beer-Lambert Law - Basic Spectrophotometric Procedure (optional) To construct two standard curves (using linear regression model) - The importance of a standard curve - Standard curve for ONP assay - Standard curve for Bradford assay Spectrophotometric Quantification
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    4. Measuring a blank: Place the blank in the cell holder. Close the lid. Press Measure Blank to measure the blank. When the instrument is finished measuring the absorbance of the blank, the message disappears. Visible (400–800 nm) region: Standard Vis cuvet: ridges on two sides. Micro Vis cuvet : ridges on two sides (arrow sign facing the user) UV (150–400 nm) region: Standard UV cuvet : clear on all sides Micro UV cuvet : clear on all sides (arrow sign facing the user) 5. Measuring a sample: After blanking the instrument, remove the blank and place the sample in the cell holder. Close the lid. The absorbance measurement appears on the display.   Basic Spectrophotometer Operation Procedure, cont
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    Spectrophotometry determines the amount of light absorbed by colored compounds. Biochemistry is all about using spectrophotometry to measure concentration, activity, kinetics and etc. Quantitative Measurement: Beer’s Law: A = ε lc A: absorbance (no unit) : extinction coefficient (L/mole*cm) l: path length (1cm) c: concentration of compound (mole/L) Spectrophotometry
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    Why Do Many Biochemical Assays Need Standard Curves? a) To determine the concentration of substances b) To verify the linearity of assays
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    o-NP Standard Curve: To determine the amount of β - galactosidase in later experiments. Lactose Galactose + Glucose ONPG  Galactose + o-NP  (o-Nitrophenol) (O-Nitrophenyl-  , d-galactopyranoside) (colorless)      (bright Yellow,  420 nm)   -galactosidase Note: Rate of production of o-NP from ONGP is directly proportional to the  amount of  -gal  in solution only within  the linear range Standard Curve for o-NP Assay
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    Standard Curve for o-NP Assay, cont 1. Prepare samples of known ONP concentrations (Table 1). Test tube # 1 2 3 4 5 6 7 8 Volume of stock o-NP  (ml) 0 0.2 0.4 0.6 0.8 1.0 1.2 1.6 Volume of O-NP  Diluents (ml) 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.4 Final volume (ml) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Final o-NP concentration  ( μ g/ml) 0 5 10 15 20 25 30 40 2. Measure absorbance value of known O-NP concentration at 420 nm . a) blank spec with DI water, b) rinse cuvet with methanol and DI water between samples, c) record absorbance values in lab notebook. 3. Plot A 420 (x-axis) vs. O-NP concentration ( μ g/ml , y-axis) and generate a standard curve.
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    Bradford assay is widely used to measure total protein concentration in a sample solution. The binding of the dye Coomassie Brilliant Blue G-250 to protein (arginine and aromatic residues) causes a shift in the absorption maximum of the dye from 465 nm to 595 nm in acidic solutions.
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330 lab 2007 spring B part - Spectrophotometric...

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