Size Exclusion Chromatography

Practice: Which of the following statements is false?

Practice: A new protein of unknown structure has been purified & gel filtration chromatography reveals that the native protein has a molecular weight of 240 kDa. Chromatography in the presence of 6 M guanidine hydrochloride (GuHCl), a chaotropic agent that has a similar effect on proteins as urea, yields a single absorbance peak corresponding to a protein of M_r 60 kDa. Chromatography in the presence both of 6 M guanidine hydrochloride and 10 mM β-mercaptoethanol (β-ME) yields peaks for proteins of M_r 34 kDa and 26 kDa. Using this data, which option best describes the structure of this protein? Hint: sketch a visual of the protein after each chemical treatment.

Practice: To answer the questions A, B & C below, use the provided chart with the properties of the four proteins.

A) What is the order of elution of the proteins from a size-exclusion chromatography column?

     a. A → B → C → D.

     b. D → B → A → C.

     c. B → D → A → C.

     d. C → A → D → B.

B) Which pH is best for separating the proteins using anion-exchange chromatography?

     a. pH = 6.                        b. pH = 7.                  c. pH = 8.

C) In what order would the proteins elute from the anion-exchange chromatography column?

     a. A → C → D → B.

     b. D → A → B → C.

     c. B → D → C → A.

     d. C → B → D → A.