Question

What would have happened if we had adjusted the pH of our IDH extract to 4.5 and then loaded it onto the DEAE resin that was equilibrated in an IEC buffer also adjusted to pH 4.5? Explain

Answer 1

Isoelectric pH is a point at which the number of the negative charges in a protein are equal to the number of the positive charges. Therefore, the protein has not net charge and is therefore neutral. The protein has a net negative charge at a pH higher than the isoelectric pH and the protein has a net positive charge at a pH less than the isoelectric pH of the protein. In this experiment the isoelectric point of IDH protein is 8.08. The IEC (ion-exchange chromatography) buffer has pH=4.5 and the pH of the IDH is also adjusted to pH=4.5. As this pH is below the isoelectric point of this protein therefore the net charge on the protein will be positive. DEAE resin contains amino group and is thus positively charged. DEAE resin is an anion exchanger where the negatively charged proteins will bind and get eluted from the column based on the strength of the interaction between the positive charge on DEAE resin and the negative charge on the protein at a specific pH. If the pH of the IDH extract is 4.5 much below the isoelectric pH of this protein, the net charge of this protein will be positive so it will not bind to the DEAE resin which is also positively charged. In order to purify this protein the pH of the protein should be adjusted to a value more than the isoelectric pH so that the protein develops a negative overall charge and binds to the DEAE resin and can be eluted by washing it in the different salt gradient of KCl salt.