Lab 7: PRELAB
PRELAB ASSIGNMENT
- What will you be doing in lab? Why?
- What should you end up with at the end of the lab period? Where will it be located?
- Are you purifying wt or mutant protein? You will need to know the extinction coefficient of your purified protein to calculate its concentration. The extinction coefficient for HCAII is 50,420 cm-1M-1. The extinction coefficient will be different for certain mutants. Do you have such a mutation? If so, you need to calculate its extinction coefficient.
PRELAB QUIZ
Question 1: Which buffer will you use to equilibrate, wash, and elute during lab 7?
Buffer 1: 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole
Buffer 2: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole
Buffer 3: 50 mM NaH2PO4, 300 mM NaCl
HINT: Thinking about the purpose of each step will help determine which buffer should be used:
- Equilibration: the purpose of this step is to prepare the Ni-NTA resin in a buffer similar to what you will wash and elute with, so that the HCAII will optimally bind
- Wash: the goal of washing is to remove proteins that might have a small affinity for the Nickel resin (such as those with several naturally occurring histidines). Therefore the wash buffer must remove these proteins without removing HCAII.
- Elution: the goal of elution is to remove HCAII from the column so that you can collect it as it flows off.
Question 2: Connect the protein purification steps covered in class to what we are doing in lab:
How do we lyse cells in the lab?
What bulk purification step do we conduct in lab?
What fine purification step do we use?
Link to quiz (will open in a new tab): Prelab 7 Quiz