Ion-exchange chromatography basic principles and application
Answers
Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis ofdifferences in charge properties. Its large sample-handling capacity, broad applicability (particularly toproteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automationhave led to it becoming one of the most versatile and widely used of all liquid chromatography (LC)techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria forselecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purifya serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparationthrough to partial purification of the target enzyme using anion-exchange chromatography. Protocolsfor assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described.The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme withendopeptidase activity towards peptide substrates of #30 amino acids. POP is a ubiquitous post-prolinecleaving enzyme with particularly high expression levels in the mammalian brain, where it participatesin the metabolism of neuroactive
Answer:
Explanation:
Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis ofdifferences in charge properties. Its large sample-handling capacity, broad applicability (particularly toproteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automationhave led to it becoming one of the most versatile and widely used of all liquid chromatography (LC)techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria forselecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purifya serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparationthrough to partial purification of the target enzyme using anion-exchange chromatography. Protocolsfor assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described.The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme withendopeptidase activity towards peptide substrates of #30 amino acids. POP is a ubiquitous post-prolinecleaving enzyme with particularly high expression levels in the mammalian brain, where it participatesin the metabolism of neuroactive