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Related Concept Videos

Affinity and Avidity01:41

Affinity and Avidity

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Overview
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Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
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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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In a multistep reaction mechanism, one of the elementary steps progresses significantly slower than the others. This slowest step is called the rate-limiting step (or rate-determining step). A reaction cannot proceed faster than its slowest step, and hence, the rate-determining step limits the overall reaction rate.
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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: Feb 13, 2026

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
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A two-step purification strategy using calmodulin as an affinity tag.

Lianyun Lin1, Chen Liu2, Bidhan Chandra Nayak2

  • 1State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China; Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China; Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fuzhou, 350002, China; Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.

Journal of Chromatography. A
|March 4, 2018
PubMed
Summary
This summary is machine-generated.

A novel Ca2+-dependent protein purification strategy uses calmodulin (CaM) as an affinity tag. This method achieves high purity and yield for target proteins, enabling further structural and functional studies.

Keywords:
Affinity purificationCalmodulinCamodulin-binding domainGreen fluorescent protein

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Purification

Background:

  • Calmodulin (CaM) is a calcium (Ca2+)-binding protein crucial for cellular signaling.
  • CaM regulates target proteins via Ca2+-dependent conformational and hydrophobic changes, altering its binding interactions.

Purpose of the Study:

  • To develop a novel, two-step orthogonal purification strategy utilizing CaM as an affinity tag.
  • To demonstrate the method's efficacy using green fluorescent protein (GFP) as a model.

Main Methods:

  • CaM-tagging of target proteins.
  • Sequential affinity purification using immobilized CaM-binding domains.
  • Hydrophobic interaction chromatography (HIC) using phenyl sepharose.
  • Ca2+-dependent binding and ethylenediaminetetraacetic acid (EDTA)-induced elution.

Main Results:

  • High yield and purity of the purified target protein (GFP) were achieved.
  • The purified GFP retained its proper biological function.
  • The purification strategy proved effective and applicable to various protein targets.

Conclusions:

  • The novel CaM-tagging strategy offers an efficient method for protein purification.
  • This technique is valuable for obtaining functional proteins for structural and functional analyses.
  • The method's orthogonal nature and Ca2+-dependency provide precise control over purification.