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11 Nov 2019
Question I Consider the table of four proteins below: ProteinSize pl Size exclusion Charge at pHDEAE-elution 7.5 112 kDa 85 kDa 48 kDa 89 kDa 7.0 4.2 8.7 6.8 (a) In what order would the proteins elute from a size-exclusion column? (from 1- fastest to 4 = slowest). (b) Based on the pl of each protein, fill out the column in the table for charge at pH (c) What proteins have the same charge as the column at plH 7.5 and so will pass (d) In what order would the remaining proteins elute from a DEAE anion exchange (e) Now, lead yourself in steps (b) - (d) again, using a CM cation exchange column (0) To completely purify all proteins from each other, you plan to run a DEAE 7.5. Use these symbols: ++, +,.. Give a protein a double charge if its pl is more than 1 pH unit from the pH of the solution. through the column very quickly (all together, in the "void" volume)? Label those as "1" in the elution. column when you wash with high concentration NaCI? Hint: lower charged proteins will tend to come off more easily faster off. where the initial binding is at pH 5.5 followed by a wash with high concentration NaCl. column followed by a CM column. Your boss asks you to reconsider. What is wrong with your plan? What should you do instead?
Question I Consider the table of four proteins below: ProteinSize pl Size exclusion Charge at pHDEAE-elution 7.5 112 kDa 85 kDa 48 kDa 89 kDa 7.0 4.2 8.7 6.8 (a) In what order would the proteins elute from a size-exclusion column? (from 1- fastest to 4 = slowest). (b) Based on the pl of each protein, fill out the column in the table for charge at pH (c) What proteins have the same charge as the column at plH 7.5 and so will pass (d) In what order would the remaining proteins elute from a DEAE anion exchange (e) Now, lead yourself in steps (b) - (d) again, using a CM cation exchange column (0) To completely purify all proteins from each other, you plan to run a DEAE 7.5. Use these symbols: ++, +,.. Give a protein a double charge if its pl is more than 1 pH unit from the pH of the solution. through the column very quickly (all together, in the "void" volume)? Label those as "1" in the elution. column when you wash with high concentration NaCI? Hint: lower charged proteins will tend to come off more easily faster off. where the initial binding is at pH 5.5 followed by a wash with high concentration NaCl. column followed by a CM column. Your boss asks you to reconsider. What is wrong with your plan? What should you do instead?
Tod ThielLv2
7 May 2019