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Related Concept Videos

High-Performance Liquid Chromatography: Introduction01:11

High-Performance Liquid Chromatography: Introduction

High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, identify, and quantify components in complex mixtures. The term "high pressure" refers to using high pressure to push the liquid mobile phase through the tightly packed columns.
In HPLC, two phases play a critical role in the separation process:
High-Performance Liquid Chromatography: Instrumentation00:57

High-Performance Liquid Chromatography: Instrumentation

High-performance liquid chromatography, or HPLC, is an analytical technique that separates liquid samples under high pressures. An HPLC instrument consists of glass bottles for storing solvents called mobile phase reservoirs. HPLC-grade solvents are used to maintain high purity, and the dissolved gases are removed using a degasser, such as a vacuum pumping system or sparging with helium. The solvents are then pumped into the analytical column using a screw-driven syringe or reciprocating pumps.
Types Of Column Chromatography01:29

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography
When the...
Protein Folding01:22

Protein Folding

Overview
Protein Folding01:22

Protein Folding

Overview
Protein Folding01:25

Protein Folding

Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation, critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.
Protein Structure Is Critical to Its Biological Function
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing...

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Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography
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Protein folding liquid chromatography.

Quan Bai1, Xindu Geng

  • 1Key Laboratory of Modern Separation Science in Shaanxi Province, Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Northwest University, Xi'an, China. baiquan@nwu.edu.cn

Methods in Molecular Biology (Clifton, N.J.)
|December 3, 2010
PubMed
Summary
This summary is machine-generated.

This study introduces protein folding with simultaneous separation using protein folding liquid chromatography (PFLC). This method achieves high purity and bioactivity for recombinant human interferon-gamma in just one hour.

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Area of Science:

  • Biochemistry
  • Chromatography
  • Protein Science

Background:

  • Protein misfolding and aggregation are significant challenges in biotechnology.
  • Efficient refolding and purification methods are crucial for producing active recombinant proteins.

Purpose of the Study:

  • To describe a novel method for protein folding combined with simultaneous separation.
  • To introduce a two-dimensional (2D) chromatographic column for enhanced Protein Folding Liquid Chromatography (PFLC).

Main Methods:

  • Protein Folding Liquid Chromatography (PFLC) using denaturants like urea or guanidine hydrochloride.
  • Utilizing a 2D column capable of weak cation exchange or hydrophobic interaction chromatography.
  • Developing a protocol for rapid renaturation and purification of inclusion body proteins.

Main Results:

  • Achieved simultaneous protein folding and separation using PFLC.
  • Demonstrated successful application of the 2D column in PFLC.
  • Obtained recombinant human interferon-gamma with purity ≥95% and high specific bioactivity in a single step.
  • Completed the process in 1 hour.

Conclusions:

  • PFLC offers an efficient approach for protein refolding and purification.
  • The 2D column enhances the versatility and effectiveness of PFLC.
  • This single-step, rapid protocol is highly beneficial for producing active recombinant proteins.