Question

16) At which pH values would two different peptides, one with a pl of 5.9 and the other with a pl of 9.1, both bind to a cation exchange column? A) pH 3.9 B) pH 7.3 C) pH 10.9 D) None of the above 17) Which type of gel matrix (aka resin) would separate the following proteins using size exclusion chromatography: protein 1 (2,000 Da), protein 2 (7,000 Da), protein 3 (20,000 Da), and protein 4 (90,000 Da)? A) Gel of range 2,500-85,000 Da B) Gel of range 8,000-200,000 Da C) Gel of range 3,000-200,000 Da D) None of the above 18) According to the following table, which purfication step was most effective according to a calculated specific activity? Step Affinity chromatography 2,119 Anion-exchange chromatography Size exclusion chromatography Total protein (mg)Total Activity (units) 1,515 90 58,300 31,560 95,000 A) Affinity chromatography, 0.36 unitsmg B) Anion-exchange chromatography, 0.048 units/mg C) Anion-exchange chromatography, 20.8 units/mg D) Size exclusion chromatography, 0.00095 units/mg E) Size exclusion chromatography, 1055 units/mg 19) Referencing the table above, which step would be the best to omit in order to improve your purification strategy? A) Affinity chromatography B) Anion-exchange chromatography C) Size exclusion chromatography D) All columns increased the specific activity.

Answer 1

16) A) pH =3.9

Cation exchange column is having –vely charged matrix which exchanges or attracts +vely charged molecules and the molecules above their pI are –vely charged and below their pI are +vely charged, in the question we need +vely charged molecules hence the sutable pH is 3.9 where both molecules exist in +ve charge   

17) A)

Because the proteins are in the range of 2000-7000-20000 and 90000, 90k protein comes in void volume and the first to get elute followed by 20k, 7k and the last one 2k

18. E) Size exclusion chromatography because it showed 1055 activity units/mg protein

Because spf. Activity = number of units/ protein in mg

Hence 58300/2119 = 27.81; 31560/1515 =20.83; 95000/90 = 1055

19. C) Size exclusion chromatography which yielded 0.00095 units/mg