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Affinity Chromatography

Affinity Chromatography

Affinity chromatography is a powerful technique extensively utilized for separating and purifying specific biomolecules from complex mixtures. It capitalizes on the highly selective binding between an analyte and its counterpart, such as antibody-antigen interactions. The counterpart is immobilized on the stationary phase, forming an affinity column. The stationary phase typically consists of solid support, such as agarose or porous glass beads, immobilizing the affinity ligand. The mobile...

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Related Experiment Video

Updated: May 17, 2026

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Published on: April 5, 2024

Avidity-mediated virus separation using a hyperthermophilic affinity ligand.

Mahmud Hussain¹, Dustin Lockney, Ruqi Wang

¹Dept. of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.

Biotechnology Progress

|November 6, 2012

Summary

This study introduces a novel method for separating large viruses like Red clover necrotic mosaic virus (RCNMV) using a reversible binding strategy. This approach avoids harsh conditions, enabling efficient purification of viral particles.

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Last Updated: May 17, 2026

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

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Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

Published on: October 27, 2019

Area of Science:

Biotechnology
Biochemistry
Molecular Biology

Background:

Immunoaffinity separation of large, multivalent species like viruses is challenging due to strong binding requiring harsh elution.
Existing methods often necessitate stringent conditions that can damage the target molecules or the affinity ligand.

Purpose of the Study:

To develop an alternative strategy for capturing and releasing large multivalent species, specifically Red clover necrotic mosaic virus (RCNMV).
To overcome the limitations of harsh elution conditions in affinity-based separation techniques.

Main Methods:

Generated an RCNMV binding protein (RBP) with moderate affinity via Sso7d mutagenesis.
Captured RCNMV using RBP immobilized on a nickel surface via a hexahistidine (6xHis) tag, leveraging avidity.
Released RCNMV by eluting the RBP from the nickel surface, followed by size-based separation.

Main Results:

Successfully captured and released RCNMV without harsh chemical treatments.
Demonstrated a separation strategy that bypasses direct chemical conjugation of the affinity ligand.
Showcased the potential of engineered hyperthermophilic proteins as stable, non-antibody affinity ligands.

Conclusions:

The developed strategy effectively separates large multivalent species like RCNMV by harnessing avidity and reversible ligand elution.
This method offers a gentler alternative to traditional affinity separation, preserving the integrity of target molecules.
The approach is broadly applicable for purifying large molecules from complex mixtures where harsh elution is undesirable.