Your research project requires that you purify protein A away from proteins B an
ID: 175405 • Letter: Y
Question
Your research project requires that you purify protein A away from proteins B and C using a combination of gel filtration and ion exchange chromatography. Describe a purification scheme using just these two columns separately that would accomplish your goal with the least amount of protein dilution. Include in your answer the expected order of elution of the proteins from the two columns, given the physical properties of the proteins in the following table. Also justify your answer giving the scientific reasons behind your scheme.Explanation / Answer
In the situation where a salt gradient is chosen, one would usually start with a buffer of low NaCl concentration (i.e. low ionic strength), and then progressively introduce a buffer with a greater and greater salt concentration. The Na+ or Cl- ions will elute proteins from the column by neutralizing the negative or positive charges on the proteins which interact with the resin.
In this case we should setup first ion exchange chromatography, since the given isoelectric points of the proteins A, B and C are 4.2, 4.4 and 8.0 respectively. We should set up the chromatography with low ionic strength and introduce an buffer which will increase the ionic concentration gradually, to achieve this add buffer with high salt concentration. as Na+ and Cl- ions will make proteins to elute by neutralizing the charges on the protein upon interaction with resin.
As a result proteins are eluted basing in the function of the charge. Protein A and B will be eluted at both together with low positive charges will require Cl- less and thus lower Nacl concentration solution. After this upo increasing the salt concentration protein C will elute as it has high isoeletric point of 8.
Now we had separated protein c, since both the protein A and B has almost similar isoelectric point this method won’t help in separating them. However the molecular mass is of great difference, protein A is 40KDa and Protein B is 70 kDa. This difference in mass can be help for our advantage in separation. Using gel filtration these proteins will be separated. Since gel filtration works on the principle basing on size and mass itself. We can collect first protein B and then protein A will be collected at the last.
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