Problem:

Two proteins (X and Y) differ significantly in their pI values derived based on their amino acid sequence. Protein X has a pI of 8 and protein Y has a pI of 4.

Required:

Question 1: What is the functional definition of pI.

Question 2: Based on the difference in pI, state which chromatography technique you would choose to attempt to purify protein X from a mixture of proteins X and Y and briefly explain why.

Question 3: Describe the key features of a purification procedure you could use that would result in retention of protein X on the column followed by its elution. Include in your answer the sequence of steps, the buffer conditions (pH, salt
concentration) at the different stages of the procedure and the reasons you would choose these conditions.

Question 4: Why might the experiment not work as you anticipate?