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Related Concept Videos

Affinity Chromatography01:03

Affinity Chromatography

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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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Immunoprecipitation01:20

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Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
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Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Related Experiment Video

Updated: Nov 15, 2025

Protein Complex Affinity Capture from Cryomilled Mammalian Cells
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Cleavable Affinity Purification (Cl-AP): A One-step Procedure to Affinity Purify Protein Complexes.

Ammarah Tariq1, Lucy Green1, Christian Soeller1

  • 1Living Systems Institute, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.

Bio-Protocol
|March 4, 2021
PubMed
Summary

Cleavable Affinity Purification (Cl-AP) offers a novel one-step method to isolate intact protein complexes from cell lysates. This technique enables advanced biophysical and microscopy studies of tagged proteins and their interactions.

Keywords:
Affinity purificationCell biologyDrosophila melanogasterGFP-tagMitosis

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Related Experiment Videos

Last Updated: Nov 15, 2025

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Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling
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2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes
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2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Traditional methods for protein complex isolation face limitations.
  • Studying intact protein complexes is crucial for understanding cellular processes.

Purpose of the Study:

  • To introduce and validate Cleavable Affinity Purification (Cl-AP) as a streamlined technique.
  • To enable the isolation of intact fluorescent protein complexes for downstream assays.

Main Methods:

  • Utilized a tripartite system involving Protein-A-Streptavidin beads and nanobodies.
  • Employed a biotinylated, thiol-cleavable linker for controlled release.
  • Applied Cl-AP to purify GFP- and mCherry-tagged proteins and interactors from Drosophila melanogaster embryos.

Main Results:

  • Successfully purified intact GFP- and mCherry-tagged protein complexes.
  • Demonstrated the utility of Cl-AP for isolating proteins and their interacting partners.
  • Showcased the isolation of fluorescent protein complexes suitable for biophysical and microscopy assays.

Conclusions:

  • Cl-AP provides an efficient one-step purification method for tagged proteins and complexes.
  • The technique overcomes limitations of traditional reductionist approaches.
  • Cl-AP is adaptable for various antibody-nanobody combinations and protein tags.