Prelab Questions for Day 1 due at the start of the lab period: This information
ID: 212572 • Letter: P
Question
Prelab Questions for Day 1 due at the start of the lab period: This information must be in your lab notebook prior to starting lab work. You should show this to your TA before you begin. 1. What type of ion exchange media is QAE-sephadex, is it anion exchange or cation exchange? What is the ligand on the surface of the resin and how does pH affect the charge state of this group? 2. Ion exchange chromatography is highly dependent upon the pl of your protein and the pH of the elution buffer. What is a pI? The pI for your enzyme is -~9.1. Rationalize why we are using the buffer with the pH that we do to elute the protein (pH -7.5; consider the info in Chap. 5 and the Ross and Boulton paper). What is the purpose of the 2 M NaCl elution step? What do you expect to elute from the column in each step? Nitrocefin (yellow) COONa 3. Provide the chemical mechanism for the transformation of NO2 nitrocefin (yellow) into the product of the catalytic reaction (red). Hint: an active site serine is involved. NO2Explanation / Answer
QAE is an strong ion exchanger hence onces it titrated against the HCl or hyedrochloric acid then it is stable quite a wide range of pH. It does not looses it charge, therefore known as strong ion exchanger media.
2. Isoelectric pH is the pH where protein having net zero charge. Due to no negative charge at a particular pH protein does not interact with any ion excahger. Below the pI protein contain net negative charge because their carboxylic amino acids get ionized and they release the proton. Hence at low pH this protein can be purifying. It can be elute by applying the gradient of NaCl here as the concentration of Cl- ions increases than it creates an competitive environment for the negatively charged ion, hence, Cl- will replace the bind negative charge and protein will elute.
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