Exp_4_KEY_S11 - 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 absolutely selective for NADH (while 260 nm is the most sensitive for both NADH and NAD + , but not at all selective) 2. Complete the purification summary Table below LDH Purification Summary Data from one group (values will vary) total IU = ( Δ A/min)(10 6 μmol/mol)(0.001 ml)(vol of frac, ml)_____ (6220 M -1 cm -1 )(1 cm)(0.030 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) NOTES: the specific activity of the purified LDH from previous experiments generally ranged from ~180 IU/mg to ~360 IU/mg. At his stage of the class, reporting the results above in the summary table relies on pipetting skills, understanding the data when generated and judging whether or not to repeat an assay (eg linearity and proportionality clearly demonstrated for enzyme assays; or standard curve and dilution series of samples in Bradford reliable?), and performing the calculations correctly. 3. Complete the affinity column binding capacity Table. Briefly describe what changes you might make to
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This note was uploaded on 11/10/2011 for the course MCB 120L taught by Professor Fairclough during the Spring '08 term at UC Davis.

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Exp_4_KEY_S11 - K EY EXPERIMENT 4 1 From the overlay...

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