Question

1) describe isoelectric focusing and isopycnic (density) separation techniques 2) why are these real focusing techniques? 3) how are these gradients established in both cases? d) describe the difficulties in using each 4) why is chromatofocusing not a real focusing technique? 5) describe advantages of these techniques and give examples

Answer 1

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.

Isopycnic means "of the same density." In particular, an isopycnic surface is a surface of constant density. This term is a bit more obscure than the similar terms isobaric or isothermal surfaces, which describe surfaces of constant pressure and constant temperature respectively. It is common in conversational use to hear isopycnic surfaces referred to simply as "iso-density" surfaces, which while strictly incorrect, is nonetheless abundantly more clear.

4)why is chromatofocusing not a real focusing technique:Chromatofocusing is a protein-separation technique that allows resolution of single proteins and other ampholytes from a complex mixture according to differences in their isoelectric point. Chromatofocusing utilizes ion exchange resins and is typically performed on fast protein liquid chromatography (FPLC) or similar equipment capable of producing continuous buffer gradients though this is not a requirement. In contrast to typical ion exchange chromatography, where bound molecules are eluted from the resin by increasing the ionic strength of the buffer environment, chromatofocusing elutes bound species by altering the pH of the buffer. This changes the net surface charge of bound molecules, altering their avidity for the resin. As the changing pH of the buffer system traverses the pI of a given molecule, that molecule will elute from the resin as it will no longer possess a net surface charge (a requisite for molecular binding to ion exchange resins). Chromatofocusing is a powerful purification technique with respect to proteins as it can resolve very similar species only differing by 0.02 pH units that may not separate well, or at all, using traditional ion exchange strategies. A major drawback to this technique is that some proteins will aggregate when they are present at relatively high concentrations and carry no net surface charge. This can cause blockage of the resin, which is highly problematic when using sealed columns of ion exchange resin on FPLC equipment, resulting in pressure buildup and possible equipment failure. Apparent aggregation issues can sometimes be overcome by limiting the sample concentration and use of buffer additives that deter aggregate formation.

5)advantages:Chromatofocusing is a powerful purification technique with respect to proteins as it can resolve very similar species only differing by 0.02 pH units that may not separate well, or at all, using traditional ion exchange strategies.

chromatofocusing utilizes ion exchange resins and is typically performed on fast protein liquid chromatography (FPLC)

This changes the net surface charge of bound molecules, altering their avidity for the resin