Exp_4_Key - KEY EXPERIMENT 4 1. From the overlay spectra,...

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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 + ) 2. Complete the purification summary below, Table 4.5, using data from Tables 4.2 and 4.4. Table 4.5 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 22 960 100 24.9 38.6 1 60% pellet 0.55 575 60 12.1 47.5 1.2 Combined G-75 fractions 2 333 35 2.6 128 3.3 purified/dialyzed LDH 6 125 13 0.44 283 7.3 total IU = ( A/min)(10 6 μmol/mol)(250 x 10 -6 L)(vol of frac, ml)____________ (6220 M -1 cm -1 )(0.55 cm)(0.025 ml)(dilution, working stock)(dilution in series) % 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 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
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This note was uploaded on 04/07/2010 for the course MCB 102,103,12 taught by Professor Segel during the Spring '10 term at UC Davis.

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Exp_4_Key - KEY EXPERIMENT 4 1. From the overlay spectra,...

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