You are given a sample containing two proteins inphosphate-buffered saline: Protein A(pI value 8.4) andProtein B (pI value 7.0).
You wish to separate this protein mixture byion-exchange and have available a column of CMÂSephadex. To carryout the procedure, you have prepared an appropriate MES buffer (50mM, pH 6.0, with 20 mM NaC1). We will call this BufferM.
(i) Suggest a preparatoryprocedure and materials needed to change the phosphate buffer ofthe protein sample with the MES buffer (Buffer M)required for ion-exchange.
(ii)Once theproteins are introduced into Buffer M, comment on the overallcharge states for each of ProteinA and Protein B.
(iii)The column ofCM-Sephadex is equilibrated with Buffer M. What is the overallcharge state of the matrix under these bufferconditions?
(iv)Theprotein mixture is now introduced to the column. Describe theprocess of adsorption that will occur. Which ofthe two proteins, Protein Aor Protein B, willexperience strongest adsorption under theseconditions? Justify your answer.
(v) What should be done nextto effect the best separation of ProteinA and Protein Bfrom the matrix? Give a full moleculardescription of events.
(vi) Consider a differentseparation procedure in which a different buffer choice was made tocarry out the separation on CM-Sephadex. In this second scenario,Buffer T, containing 50 mM Tris buffer, pH 7.5with 50 mM NaC1 was used for starting conditions.Outline how a separation of ProteinA and Protein Bcan still be effected in Buffer T utilizingCM-Sephadex matrix. Explain the order of elution that wouldoccur for each of the Protein A andProtein B species with your newprocedure.
(vii) Comment on whether itwould be possible to separate Protein Afrom Protein B againusing Buffer T, but with a different column chemistry, onecontaining Mono-Q matrix.
