Concept #1: Size-Exclsuion Chromatography
Practice: In a mixture of 5 proteins (listed below), which protein elutes 2 nd in 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 Mr 60 kDa. Chromatography in the presence both of 6 M guanidine hydrochloride and 10 mM β-mercaptoethanol (β-ME) yields peaks for proteins of Mr 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.