Question

List two different ways you could change the buffer to elute the bound protein(s) and achieve proper separation of the proteins. Select all that apply. Please explain why.

add a buffer with pH=8.0

add a buffer with pH=5.0

decreasing the ionic strength of the buffer

increasing the ionic strength of the buffer

Answer 1

• Elution is a method of separation of proteins. Proteins have various groups that are potentially ionizable from the side chain of their amino acid chains.
• One of the method by which buffer can be changed for proper separation of protien is adding a buffer with pH = 8. When the pH of the buffer is equal to the PI (Isoelectric Point) of the protien, the total charge on the protien becomes zero and it can be easily separated. For this, the buffer has to maintain a basic pH of about pH = 8.
• Another method is to increase the ionic strength of the buffer. With the increase of ionic strength of buffer, the protein ions will be replaced by the buffer ions. This supports in better separation of protein as well.