Once the enzyme solution has been prepared it must be

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Unformatted text preview: on in your lab notebook. 2. Use the dilution scheme to on the next page to prepare a series of samples. 3. Determine the absorbance at 340 nm of the series of NADPH samples that you just prepared. Use Sample 1 as the blank. Blank the spectrophotometer. 4. Measure and record A340 for each sample. For each sample, record the absorbance every ~30 seconds until you have three absorbance values. Calculate the average of these three measurements. 5. On your own, plot your data and determine the molar absorptivity (ε) for NADPH. Compare your experimental value to the literature value of 6220 M- 1cm- 1. If your value is outside the range of 5000 M- 1 cm- 1 to 7000 M- 1 cm- 1, use the literature value in the data analysis. Dilution scheme: Sample Vstock NADPH, µ L Vbuffer, µ L Vtotal, µ L 1 0 2000 2000 2 1000 3000 4000 3 400 1600 2000 4 300 1700 2000 5 250 1750 2000 6 167 1833 2000 7 1667 2000 use 333 µ L of sample 2 8 1833 2000 use 167 µ L of sample 2 Part II: Making the enzyme Alcohol dehydrogenase from liver has been isolated, purified, and lyophilized (dried). Your TA will be making fresh enzyme to use in this experiment....
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