1. Protein purification in practice Quantification of the procedure –How well did it work? –Did something go wrong? Where? Know how much fumarase is present at each step –“Yield” –Total enzyme activity (  mol malate/min) –Need activity of a certain volume of the fraction and the volume of the fraction. Know how pure fumarase is at each step –“Specific activity” –Enzyme activity per mg of total protein For each major sample, record: –activity (  abs/time): specifically how much fumarase is present –protein concentration (Bradford): total indiscriminate protein –volume

2. SAMPLE 1SAMPLE 2 Which has a higher yield of fumarase? Which will show a higher specific activity? Which has a higher fold purification? Starting material

3. Major Samples Crude extract –How much fumarase did you start with? –aka “source material”, “pre-column extract”, “applied” Purified enzyme x 2 –pre- and post-dialysis) –How much did you recover?

4. Major Samples Crude extract Flow-through –How much fumarase bound to the column? –How much flowed through the column? –“Post-column extract” –What should the specific activity of the FT be? Purified enzyme

5. “Purification Table” Volume Total Protein (mg) Total Activity (  mol/min) Specific Activity (  mol/min/mg) Yield (%) Fold Purification Crude Column Flow Through Purified (Pre- dialysis) Purified (Post-dialysis) Need to know mg/mL (Bradford)

6. General procedure Prepare column –“Flow adaptor” –No air –Wash column briefly with extraction buffer Prepare sample –Re-centrifuge Start collecting fractions! Apply sample to column Wash column with extraction buffer Elute fumarase with several “column volumes” of elution buffer (imidazole) –Collect smaller fractions (~1.5 mL)

7. Measure A280s of fractions A280 Fraction number Flow Through “Wash” Start elution (500 mM Imidazole) Wash: remove all low-affinity proteins from the column A280 should return ~ baseline Elute: Protein (fumarase) should be collected A280 ≠ protein here

8. Imidazole elutions Contain fumarase (theoretically) Contain ~500 mM Imidazole –May inhibit fumarase activity Give falsely low reading for yield, specific activity? “Dialyze” the protein –Lower the imidazole concentration

9. Dialysis of biological molecules Not really a purification procedure –Removes salts & other small molecules –Not designed to remove other proteins Place protein solution in a “bag” made from a semipermeable membrane –Permeable to water and small molecules Place dialysis bag in a container with lower salt buffer –Diffusion/osmosis for system to achieve osmotic balance Salts/small molecules diffuse out Water diffuses in

10. Beginning dialysisAfter equilibration

11. Dialysis ~ Dilution Start with 3 mL of fumarase with 500 mM Imidazole Dialyze overnight in 500 mL of dialysis buffer (no Imidazole) What’s the final concentration of Imidazole in the sample?

12. Beginning dialysisAfter equilibration 3 mL 500 mM Imidazole ? 500 mL

13. Fractions you’ll have Crude (stored at 4°) (save a few hundred  L) Crude stored frozen Flow through (pool from fraction collector) Eluted fumarase –Assays to determine which fractions contain fumarase –Pool fumarase –Save a few hundred  L –Dialyze the rest Dialyzed fumarase