Question

A E A A A * A.A E 21 te - Аавьсера лавьсера Аавьс Аавьссс Аав Аавьссо Аавьсара | Normal 1 No Spac... Heading 1 Heading 2 Title Subtitle Subtle Em... Paragraph Styles 4. Be able to differentiate what type of column you can purify your protein based on the PI of protein, the pH of stability, and the goal of binding protein to type of beads. (an example, your protein has a Pl of 5.1 with a stability of pH range 3.9 to 5.5, what pH and column type (anion/cation) would you run the protein in order for it to bind the column, and how would you elute (remove) it off the column)?

Answer 1

The given protein can be purified by Ion Exchange Chromatography.
Ion exchange protein purification can be achieved as most of the proteins possess nonzero net electrostatic charges at every pH value except the pH value is equal to pI value i.e (pH=pI). When the pH>pI of a protein to be tested, that protein becomes negatively charged. Hence cationic column have been used, where the pH<pI of the same protein to be tested, it becomes positively charged, henceforth anionic column have been used.
The basic principle behind Ion exchange chromatography is that it happens due to electrostatic attraction between the charged proteins dissolved in the buffer and the oppositely charged binding sites on a solid adsorbent (Ion exchange). i.e an ion exchange adsorbent is otherwise known as media, resin, a matrix or a gel which usually consists of a spherically porous beads with functionally charged groups which was densely grafted onto the surface of the beads. Hence the functional group charges are neutralized by the free counter –ions.
The procedure to be followed in the purification of proteins in ion-exchange chromatography is
•   The mixture of protein is transferred to low ionic strength buffer ie it is considered as a mobile phase.
•   The ion exchange adsorbent (stationary phase) is packed into the column which is pre- equilibrated with the buffer of identical pH and the same ionic strength as the protein mixture.
•   When the mixture of protein applied onto the column. The proteins which are charged oppositely to the ion-exchange media is temporarily retained in the column.
•   Then the retained proteins are eluted from the column by applying a modified buffer solution. In general the process of elution was achieved by gradual increase of ionic strength of the buffer solution through salt gradient. The proteins are further eluted in order to increase their net charge. In particular cases the process of elution can be accomplished by the change in pH and other affinity methods.