Exp_4_Key_W10 - K EY EXPERIMENT 4 1. From the overlay...

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KEY EXPERIMENT 4 1. From the overlay spectra, briefly discuss why 340 nm was used for the LDH enzyme activity. 340 nm is selective for NADH (while 260 nm is the most sensitive for both NADH and NAD + ) 2. Complete the purification summary below, Table 4.5, using data from Tables 4.2 and 4.4. Table 4.6A LDH Purification Summary Data from one group (values will vary) Fractionation Step Fraction ml Total IU % Yield of Total IU Total mg Specific activity IU mg -l Fold purification Crude extract 24 2844 100 82.9 34.3 1 40% supernatant 24 2670 94 64.8 41.2 1.2 60% pellet 5.2 1823 64 31.2 58.4 1.7 purified/dialyzed LDH 1.5 849.4 30 1.1 773 22.5 total IU = ( Δ A/min)(10 6 μmol/mol)(0.001 L)(vol of frac, ml)_ (6220 M -1 cm -1 )(0.03 ml)(dilution) % yield = (total IU of the step) / (total IU of the crude extract) total mg = (mg/ml from Bradford protein assay)(vol of frac, ml) specific activity = total IU / total mg fold-purification = (specific activity of a step) / (specific activity of the crude extract) 3. Complete the affinity column binding capacity Table 4.7A below. Briefly describe what changes you might make to increase the capacity of the affinity column to bind LDH ( ie improve the over-all yield of LDH purified by using an affinity column). Table 4.7A
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Exp_4_Key_W10 - K EY EXPERIMENT 4 1. From the overlay...

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