Question

You have ion exchange, gel filtration and NAD-affinity columns available to you. Which ones would you use to purify protein A away from the other three proteins? Sketch the elution curve (A280 VS elution volume) you would get if all 4 proteins were run on (A) a gel filtration column, (B) an anion exchange column at pH7 and eluted with a gradient of increasing NaCl concentration and (C) an NAD affinity column and eluted with a gradient of increasing NAD concentration.

Protein     pI        Molecular Weight      Affinity for NAD

A            5           30,000                      ++++

B            8          60.000                      ++

C            5           60,000                       -

D            8           30,000                       -

Answer 1

a) Principle behind gel filtration column is that separation of protein is based on its size, shape or weight. porous resin is selected so that small molecules from the mixture can pass and large molecules can be excluded and elute first as compared to smaller molecules. in the given case protein A and protein B elute first.

b) Principle behind an anion exchange column is that separation of molecules takes place on the basis of surface charge. in this case resin has + charge and molecule of interest having negative charge binds to it and elute later as compared to positive charged molecule. Nacl concentration is used to separate the bound molecule. pH of the buffer is 7, so the proteins having pH extremely below the pI contains strong positive charge on it and can bind both weak and strong cation resin. in the given case, protein D contain strong positive charge and do not bind to anion resin, therefore elute first then protein A. protein A has significantly high pI then pH of the buffer that is why it contains strong negative charge on it.

c) principle of an affinity column involves the process of entrapment in which target molecule get traped with the stationary phase. in this case target molecule having more affinity for NAD get trapped and elute later. therefore, protein A elute at last as compared to other 3.

to purify protein A we can use both ion exchange and gel filtration to compleately purify it. in size exclusion we can separate protein A and D because of their same molecular weight. then by using ion exchange we can easily separate bothnof the because both have different pI value.