Your first project in your new lab is to purify the protein Rock Band (RB) which is known to be phosphorylated by the kinase Heavy Metal Drummer (HMD). As the new kid on the block, you are tasked with purifying tons of HMD for the lab to use. You first attempt to purify the kinase using ion-exchange chromatography. You scour PubMed and determine that the isoelectric point (pI) of HMD is pH 5.5.

(i) What should the pH of your buffer be if you wanted to bind HMD to an anionexchange column? Why?

(ii) What should the pH of your buffer be if you want to bind HMD to a cation-exchange column? Why?

(iii) What potential issues might arise if the pH of your buffer is too close to the pI of your protein of interest? Why might an extreme buffer pH (< 6 or > 9) be unsuitable?