BACE Chromatography Techniques 2

Question 1

Which chromatography technique is most commonly used to purify His-tagged recombinant proteins?

Accepted Answer

Immobilized metal affinity chromatography (IMAC)

Answer

IMAC uses a metal ion (e.g., Ni²⁺) chelated to the resin that binds specifically to the polyhistidine (His) tag on recombinant proteins.

Question 2

In reverse-phase HPLC, the stationary phase is:

Accepted Answer

Nonpolar and hydrophobic

Answer

Reverse-phase HPLC uses a nonpolar, hydrophobic stationary phase (e.g., C18-bonded silica) that retains hydrophobic analytes.

Question 3

What reagent is typically used to elute a protein bound to a Ni-NTA IMAC column?

Accepted Answer

Imidazole at high concentration

Answer

Imidazole competes with the histidine tag for binding to the Ni²⁺ ion, displacing the His-tagged protein and causing it to elute.

Question 4

What does 'void volume' mean in the context of size exclusion chromatography?

Accepted Answer

The volume at which the largest excluded molecules elute

Answer

The void volume (V₀) is the volume of mobile phase outside the pores; molecules too large to enter the pores elute at this volume.

Question 5

Hydrophobic interaction chromatography (HIC) typically uses which condition to bind proteins to the resin?

Accepted Answer

High salt concentration

Answer

HIC binding is promoted by high salt (kosmotropic salts like ammonium sulfate) which enhances hydrophobic interactions between the protein and the resin.

BACE - Biotechnician Assistant Credentialing Exam Practice Test

BACE - Biotechnician Assistant Credentialing Exam Practice Test

BACE Chromatography Techniques 2