Related Experiment Video
Updated: Jun 3, 2026
07:19
Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System
Published on: April 26, 2024
Purification of recombinant proteins having high isoelectric points
1Department of Genetics and Molecular Biology, University of Napoli, Italy.
Methods in Molecular Medicine
|March 11, 2011
Summary
Recombinant antigens and synthetic peptides offer sensitive diagnostic assays, especially in virology, by avoiding hazardous materials. Protein purification is crucial and depends on antigen epitopes and expression systems.
Area of Science:
- Biochemistry
- Immunology
- Virology
Background:
- Recombinant antigens and synthetic peptides are advanced tools for developing sensitive diagnostic assays.
- In virology, recombinant antigens reduce the need for handling hazardous materials and enable multiepitope polypeptide assembly.
Purpose of the Study:
- To highlight the critical role of protein purification in preparing recombinant antigens.
- To discuss factors influencing recombinant antigen purification and their impact on assay reliability.
Main Methods:
- Discusses the biochemical characteristics of recombinant proteins.
- Analyzes the dependence of purification strategies on immunodominant epitopes (continuous and discontinuous).
- Considers the impact of expression systems, conditions, proteases, and purification steps on yield and purity.
Main Results:
- Recombinant protein characteristics vary based on expression systems and conditions.
- Epitope accessibility and type (continuous/discontinuous) dictate purification challenges.
- Expression system, proteases, and purification steps significantly affect antigen yield and purity.
Conclusions:
- Effective purification of recombinant antigens is essential for reliable diagnostic assays.
- Understanding epitope structure and optimizing expression/purification are key to successful recombinant antigen development.
Related Concept Videos
01:22Two-dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as cells...
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as cells...
01:20Immunoprecipitation
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
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...
01:18Detergent Purification of Membrane Proteins
Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and...
01:29Downstream Processing
Downstream processing begins once fermentation is complete and involves a series of steps to recover and purify products such as acids, vitamins, antibiotics, or proteins.Cell HarvestingFor example, for intracellular protein-based products, the first step is harvesting the cells. This is typically achieved using centrifugation or filtration to separate the cells from the liquid phase.Cell Disruption for Intracellular ProductsIf the target product is intracellular, the harvested cells must be...
01:27SDS-PAGE
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
01:29Types 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...
Gel Filtration Chromatography
When the...