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30 Aug 2018
Consider two proteins: Protein A of pI value 5.3, Protein B of pI value 7.8.
(i) You have available a column of Sepharose-Q matrix and solutions of TRIS buffer (150 mM,
pH 7.5) and sodium chloride (1M).
Write out a description of steps required to achieve separation of these proteins from one
another by ion exchange chromatography. Clearly indicate in your summary the overall
charge state of both proteins and the solid matrix. Explain which of the proteins (if any)
will be adsorbed to the matrix during your chromatography run, and how elution will be
controlled.
(ii) Comment on the relative distribution of Asp, Glu, Lys and Arg residues within the primary
sequences of Protein A and Protein B.
Consider two proteins: Protein A of pI value 5.3, Protein B of pI value 7.8.
(i) You have available a column of Sepharose-Q matrix and solutions of TRIS buffer (150 mM,
pH 7.5) and sodium chloride (1M).
Write out a description of steps required to achieve separation of these proteins from one
another by ion exchange chromatography. Clearly indicate in your summary the overall
charge state of both proteins and the solid matrix. Explain which of the proteins (if any)
will be adsorbed to the matrix during your chromatography run, and how elution will be
controlled.
(ii) Comment on the relative distribution of Asp, Glu, Lys and Arg residues within the primary
sequences of Protein A and Protein B.
Nestor RutherfordLv2
1 Sep 2018
