BACE Chromatography Techniques 2 — Questions and Answers
Question 1: Which chromatography technique is most commonly used to purify His-tagged recombinant proteins?
- Size exclusion chromatography
- Ion exchange chromatography
- Immobilized metal affinity chromatography (IMAC) (Correct answer)
- Reverse-phase HPLC
Correct answer: Immobilized metal affinity chromatography (IMAC)
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:
- Highly polar and hydrophilic
- Nonpolar and hydrophobic (Correct answer)
- Positively charged at neutral pH
- A porous gel with defined pore size
Correct answer: Nonpolar and hydrophobic
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?
- High salt (e.g., 1 M NaCl)
- Imidazole at high concentration (Correct answer)
- Low pH buffer (pH 3.0)
- Reducing agent such as DTT
Correct answer: Imidazole at high concentration
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?
- The volume of buffer needed to equilibrate the column
- The volume at which the largest excluded molecules elute (Correct answer)
- The total volume of liquid within the column
- The sample volume that can be loaded safely
Correct answer: The volume at which the largest excluded molecules elute
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?
- Low salt concentration
- High salt concentration (Correct answer)
- Low pH
- High imidazole concentration
Correct answer: High salt concentration
HIC binding is promoted by high salt (kosmotropic salts like ammonium sulfate) which enhances hydrophobic interactions between the protein and the resin.
Question 6: Which instrument component in an HPLC system is responsible for detecting separated analytes as they exit the column?
- Pump
- Injector
- Detector (Correct answer)
- Guard column
Correct answer: Detector
The detector (e.g., UV-Vis, fluorescence, or refractive index) monitors the column effluent and generates a signal proportional to analyte concentration.
Question 7: In ion exchange chromatography, increasing the salt concentration in the elution buffer causes bound proteins to:
- Bind more tightly to the resin
- Elute from the resin (Correct answer)
- Precipitate inside the column
- Migrate to a different zone on the column
Correct answer: Elute from the resin
Increasing ionic strength introduces competing ions that displace the bound protein from the charged resin, causing it to elute.
BACE Chromatography Techniques 2
Which chromatography technique is most commonly used to purify His-tagged recombinant proteins?