Question

Purification of 6×His-tagged protein using Ni-NTA column is an example of

• A. affinity chromatography
• B. hydrophobic-interaction chromatography
• C. ion-exchange chromatography
• D. size-exclusion chromatography

Hint:

Affinity chromatography is a powerful technique for purifying proteins based on specific binding interactions. The His-tag allows for efficient purification using Ni-NTA resins.

Answer 1

The Correct Option is A

Step 1: Understanding the principle of affinity chromatography.
In affinity chromatography, proteins or other molecules are purified based on their specific interactions with a ligand. The use of a 6×His tag allows for the specific binding of the protein to a nickel (Ni) or cobalt (Co) ion in the column via the imidazole group of histidine residues, facilitating the purification process. Ni-NTA (Nickel-Nitrilotriacetic acid) resin specifically binds to the His-tag, and after washing away non-bound material, the His-tagged protein is eluted using a competitive imidazole solution.
Step 2: Analyzing the options.
- (A) Affinity chromatography is the correct technique used for purifying proteins with specific tags, such as the His-tag. - (B) Hydrophobic-interaction chromatography relies on hydrophobic interactions between the protein and the column material, not on specific binding like in affinity chromatography. - (C) Ion-exchange chromatography separates proteins based on their charge, not the specific interaction with a ligand. - (D) Size-exclusion chromatography separates proteins based on their size, not specific binding interactions.
Step 3: Conclusion.
The correct answer is (A) affinity chromatography, as this technique is specifically used to purify His-tagged proteins using a Ni-NTA column.