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Related Experiment Video

Updated: Mar 15, 2026

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

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Published on: June 20, 2019

Novel NTA-Ni²⁺ Agarose-Based Microspheres: Structural Features and Chromatographic Capacity.

Min Zhao¹, Chen Liang¹, Boheng Liu¹

¹Department of Marine Pharmacology, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.

|March 14, 2026

Summary

This study optimized agarose-based microspheres for immobilized metal affinity chromatography (IMAC). The developed NTA-Ni2+ agarose-based microspheres (ABM) show high binding capacity and efficient purification of His-tagged proteins.

Keywords:

NTA-Ni2+ agarose-based microspheres immobilised metal affinity chromatography protein purification response surface method

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Published on: November 19, 2015

Area of Science:

Biotechnology
Chromatography
Materials Science

Background:

Immobilized metal affinity chromatography (IMAC) media are essential for recombinant protein purification.
Optimizing IMAC media enhances purification efficiency.

Purpose of the Study:

To design and optimize NTA-Ni2+ agarose-based microspheres (ABM) for efficient protein purification.
To establish optimal conditions for ABM preparation using a Box-Behnken design.

Main Methods:

Agarose-based microspheres prepared using a three-factorial Box-Behnken design.
One-step crosslinking of epichlorohydrin (ECH)-nitrilotriacetic acid (NTA) for ligand coupling.
Characterization using SEM, AFM, DSC, FTIR, and SDS-PAGE.

Main Results:

Optimal conditions determined: 8.12 wt% emulsifier PP, 1624.46 rpm stirring speed, 53.86 °C oil-phase temperature (span value Y=0.50684).
Structurally stable NTA-Ni2+ ABM with uniform cross-linking network confirmed by SEM, AFM, DSC, and FTIR.
High binding capacity (15.2 ± 0.8 mg/mL) for His-tagged proteins and efficient purification demonstrated by SDS-PAGE.

Conclusions:

The study provides a rational design and practical solution for IMAC medium development.
Optimized NTA-Ni2+ ABM offers enhanced performance for recombinant protein purification.