Protein Chromatography

Carta, Giorgio; Jungbauer, Alois

doi:10.1002/9783527824045

Cited by 42 publications

(24 citation statements)

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“…Therefore, the data from Figure A for the intermediate ligand density have been used. Again, the Langmuir model is most often used for Protein A materials despite different base materials, different Protein A-derived proteins, as well as the use of polyclonal or monoclonal IgG species. ,,, The Langmuir isotherm applies for monolayer adsorption and is given by , where q is the adsorbate load , C is the equilibrium adsorbate concentration, q max is the maximum capacity, and K L is the equilibrium constant. Borlido et al could better describe the heterogeneous binding of IgG on magnetic Protein A microparticles with the Freundlich isotherm rather than with the Langmuir isotherm.…”

Section: Resultsmentioning

confidence: 99%

“…Also, Salimi et al applied the Freundlich model for the equilibrium IgG binding to magnetic Protein A microspheres. The Freundlich isotherm equation is given by , with a and b being empirical constants. Moreover, the bi-Langmuir model can describe a more heterogeneous binding as well.…”

Section: Resultsmentioning

confidence: 99%

“…This makes the different binding sites on the ligand thermodynamically inhomogeneous. , Two types of binding sites are described with the bi-Langmuir isotherm. In that way, the binding of IgG to the commercial resin Mabselect SuRe could be better described than with the Langmuir model. , The bi-Langmuir isotherm equation is given by with q max, i being the binding capacity and K L being the Langmuir equilibrium constant of each binding population. The application of all of these different fits as well as the resulting binding constants are given in the SI (Figure S4 and Table S2).…”

Section: Resultsmentioning

confidence: 99%

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Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Kaveh-Baghbaderani

Allgayer

Schwaminger

et al. 2021

ACS Appl. Nano Mater.

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The demand for purified antibodies is ever-rising. This study presents a nanoparticle-based material for efficient magnetic separation of immunoglobulin G (IgG) with high binding capacity. The characteristics include: (i) Cost-effective bare iron oxide nanoparticles are used as the solid phase on which optimized protein A-based ligands are directly immobilized. An additional chemical modification or activation is not needed. (ii) Oriented immobilization of the ligands is promoted using a C-terminal peptide tag, containing amino acids with an affinity for iron oxide. (iii) The immobilized ligand consists of eight polymerized B-domains of Protein A. This affinity adsorbent for antibody capture allows a recovery of up to 418 mg IgG per gram of particle, which exceeds the state of the art of magnetic nanoparticles as well as microparticles. Particles with a lower ligand density show a higher percentage recovery of IgG, which allows for a cost-effective design of the adsorbent. Furthermore, the selectivity of the immobilized ligand is shown by means of purification of rabbit polyclonal IgG using rabbit serum.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Kaveh-Baghbaderani

Allgayer

Schwaminger

et al. 2021

ACS Appl. Nano Mater.

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“…Chromatography describes all the processes that apply a so-called stationary phase, which can be used to separate a mixture of components that are carried by a mobile phase [ 70 ]. Mainly size exclusion chromatography (SEC) is used for the separation of exosomes.…”

Section: Separation Mechanismsmentioning

confidence: 99%

Microfluidic Approaches for Affinity-Based Exosome Separation

Theel

Schwaminger²

2022

IJMS

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As a subspecies of extracellular vesicles (EVs), exosomes have provided promising results in diagnostic and theranostic applications in recent years. The nanometer-sized exosomes can be extracted by liquid biopsy from almost all body fluids, making them especially suitable for mainly non-invasive point-of-care (POC) applications. To achieve this, exosomes must first be separated from the respective biofluid. Impurities with similar properties, heterogeneity of exosome characteristics, and time-related biofouling complicate the separation. This practical review presents the state-of-the-art methods available for the separation of exosomes. Furthermore, it is shown how new separation methods can be developed. A particular focus lies on the fabrication and design of microfluidic devices using highly selective affinity separation. Due to their compactness, quick analysis time and portable form factor, these microfluidic devices are particularly suitable to deliver fast and reliable results for POC applications. For these devices, new manufacturing methods (e.g., laminating, replica molding and 3D printing) that use low-cost materials and do not require clean rooms are presented. Additionally, special flow routes and patterns that increase contact surfaces, as well as residence time, and thus improve affinity purification are displayed. Finally, various analyses are shown that can be used to evaluate the separation results of a newly developed device. Overall, this review paper provides a toolbox for developing new microfluidic affinity devices for exosome separation.

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“…While ow chromatography systems especially bene t from relatively high throughput and scalability [5,9,10], several analytical applications such as sequencing and protein crystallography require only a small amount of puri ed protein. For such applications, techniques such as polyacrylamide gel electrophoresis (PAGE) are used to separate biomolecules according to their size and net electric charge [11,12].…”

Section: Introductionmentioning

confidence: 99%

A Simple Micropreparative Gel Electrophoresis Technique for Purification of Nanoscale Materials

Sajjadi

Zubkovs

et al. 2022

Meet. Abstr.

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Many biochemical, biomedical, and material applications hinge on the ability to effectively separate and purify nanoscale materials. Though this need is largely addressed with biological macromolecules using a variety of chromatographic and electrophoretic purification techniques, such techniques are usually laborious, time-consuming, and often require complex and costly instalments that are inaccessible to most laboratories. Synthetic nanoparticles face similar purification challenges, often relying on techniques that are material-specific. In this work, we introduce a versatile micro-preparative (MP) method based on polyacrylamide gel electrophoresis (PAGE) to purify biological samples containing proteins, nucleic acids, and complex bioconjugates, as well as synthetic nanoparticles based on graphene quantum dots (GQDs). Using a conventional vertical slab PAGE, we demonstrate the extraction of purified DNA, proteins, and DNA-protein bioconjugates from their respective mixtures using MP-PAGE. We apply this system to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using specialized commercial devices. We also demonstrate the purification of folded enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, comparable to purities achieved using a two-step size exclusion and immobilized metal-ion affinity chromatography purification procedure. Moreover, we demonstrate the successful isolation of an EYFP-DNA bioconjugate that otherwise could not be processed using the two-step chromatography procedure. Finally, the technique was further extended to demonstrate size-dependent separation of a commercial mixture of GQDs into three different fractions with distinct optical properties. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

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Protein Chromatography

Cited by 42 publications

References 0 publications

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Microfluidic Approaches for Affinity-Based Exosome Separation

A Simple Micropreparative Gel Electrophoresis Technique for Purification of Nanoscale Materials

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