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Related Experiment Video

Updated: May 29, 2026

High-Throughput Protein Crystallization via Microdialysis
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High-Throughput Protein Crystallization via Microdialysis

Published on: March 3, 2023

High-throughput protein purification and quality assessment for crystallization.

Youngchang Kim1, Gyorgy Babnigg, Robert Jedrzejczak

  • 1Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.

Methods (San Diego, Calif.)
|September 13, 2011
PubMed
Summary

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High-quality protein samples are essential for structural biology. The Midwest Center for Structural Genomics (MCSG) developed semi-automated, high-throughput protocols for protein expression and purification using immobilized metal affinity chromatography (IMAC).

Area of Science:

  • Structural biology
  • Protein biochemistry
  • Genomics

Background:

  • High-quality protein samples are critical for determining protein structures using X-ray crystallography or NMR spectroscopy.
  • Achieving "structural biology-grade" proteins requires reproducible purification yielding milligram quantities of homogeneous protein.
  • Advancements in structural genomics necessitate efficient, high-throughput protein production methods.

Purpose of the Study:

  • To present semi-automated, high-throughput protocols for parallel protein expression and purification developed at the Midwest Center for Structural Genomics (MCSG).
  • To detail a two-step immobilized metal affinity chromatography (IMAC) purification strategy for generating structural biology-grade proteins.
  • To discuss the integration of these purification methods into large-scale structural genomics pipelines.

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High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography
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Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
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Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Published on: July 5, 2018

Related Experiment Videos

Last Updated: May 29, 2026

High-Throughput Protein Crystallization via Microdialysis
06:18

High-Throughput Protein Crystallization via Microdialysis

Published on: March 3, 2023

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography
06:19

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

Published on: March 10, 2023

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
08:58

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Published on: July 5, 2018

Main Methods:

  • Proteins are expressed as fusion proteins with a cleavable affinity tag.
  • A two-step immobilized metal affinity chromatography (IMAC) process is employed: IMAC-I with buffer exchange/size exclusion chromatography, followed by Tobacco Etch Virus (TEV) protease cleavage.
  • IMAC-II removes the cleaved tag and residual TEV protease, yielding purified protein.

Main Results:

  • Developed semi-automated protocols enabling high-throughput parallel protein expression and purification.
  • Successfully produced many proteins in large-scale quantities using the described two-step IMAC method.
  • These protocols are implemented on multidimensional chromatography workstations.

Conclusions:

  • The developed semi-automated protocols are effective for high-throughput production of structural biology-grade proteins.
  • The two-step IMAC purification strategy, including TEV protease cleavage, efficiently yields homogeneous protein samples.
  • These methods are crucial for advancing structural genomics initiatives like MCSG and CSGID.