Gel Filtration: Size Exclusion Chromatography

Hagel, Lars

doi:10.1002/9780470939932.ch3

Cited by 35 publications

(28 citation statements)

References 164 publications

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“…Molecules larger than the pores cannot be separated and will elute in the so called void volume. Eluted fractions are detected mainly by concentration sensitive detectors (e.g., UV/Vis or refractive index) and a linear relationship persists between elution volume and logarithm of the hydrodynamic size [128,129]. Therefore, calibration of SEC with molar mass standards is possible for analytes with similar structure (e.g., globular proteins), whereas molar mass of macromolecules with other structure (e.g., linear, branched) may be over-or underestimated because of a deviating elution behaviour.…”

Section: Separation Principlementioning

confidence: 99%

“…The column most frequently used for studying cross-linked casein is Superdex 200, e.g., [30,44,47,48,54,100,115,131,[134][135][136][137][138][139][140][141][142][143][144][145][146][147][148], which is provided by GE Healthcare (Uppsala, Sweden). The column material is based on agarose/dextran with a ratio of pore volume to void volume of V i /V o = 1.7 and exhibits a fractionation range from 10 to 600 kg/mol for globular proteins [128]. Superose 6 columns (GE Healthcare, Uppsala, Sweden) that are packed with agarose beads with V i /V o = 1.87 and exhibit a greater separation range of 5 to 5000 kg/mol for globular proteins [128] were also applied, e.g., [43,118,149].…”

Section: Separation Principlementioning

confidence: 99%

“…The column material is based on agarose/dextran with a ratio of pore volume to void volume of V i /V o = 1.7 and exhibits a fractionation range from 10 to 600 kg/mol for globular proteins [128]. Superose 6 columns (GE Healthcare, Uppsala, Sweden) that are packed with agarose beads with V i /V o = 1.87 and exhibit a greater separation range of 5 to 5000 kg/mol for globular proteins [128] were also applied, e.g., [43,118,149]. In other studies, silica-or polymer-based columns from the TSK-Gel series (Tosoh, Tokyo, Japan) were used, e.g., [91,116,132].…”

Section: Separation Principlementioning

confidence: 99%

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Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

et al. 2018

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Abstract:Casein is the major protein fraction in milk, and its cross-linking has been a topic of scientific interest for many years. Enzymatic cross-linking has huge potential to modify relevant techno-functional properties of casein, whereas non-enzymatic cross-linking occurs naturally during the storage and processing of milk and dairy products. Two size separation techniques were applied for characterisation of these reactions: gel electrophoresis and size exclusion chromatography. This review summarises their separation principles and discusses the outcome of studies on cross-linked casein from the last~20 years. Both methods, however, show limitations concerning separation range and are applied mainly under denaturing and reducing conditions. In contrast, field flow fractionation has a broad separation range and can be easily applied under native conditions. Although this method has become a powerful tool in polymer and nanoparticle analysis and was used in few studies on casein micelles, it has not yet been applied to investigate cross-linked casein. Finally, the principles and requirements for absolute molar mass determination are reviewed, which will be of increased interest in the future since suitable calibration substances for casein polymers are scarce.

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Section: Separation Principlementioning

confidence: 99%

Section: Separation Principlementioning

confidence: 99%

Section: Separation Principlementioning

confidence: 99%

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Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

et al. 2018

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“…Particles that are too large to travel through the pores will elute earlier, and molecules of smaller sizes travel through more pores and thus elute in decreasing particle size (Hagel 2011;Hagel and Janson 1992;O'Fagain et al 2011) (Figure 2.6). The volume of the peak when a particle of a certain size elutes from the column Illustration of selective pore entry based on sample particle size.…”

Section: Size Exclusion Chromatographymentioning

confidence: 99%

“…Besides particle size, elution volume is also dependent on the hydrodynamic diameter of the particles. Particles of asymmetrical shapes, such as fibrous proteins, travel faster than globular particles of the same molecular weight, appearing as if they have a larger particle size (Hagel 2011;Hagel and Janson 1992;O'Fagain et al 2011). The size of the pores and the beads in the matrix depends on the composition of the media, and media with different particle size separation range are available commercially.…”

Section: Size Exclusion Chromatographymentioning

confidence: 99%

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

Abidin¹

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This research examined the ability of virus-like particles (VLP) assembled from the coat proteins of Murine polyomavirus (MPyV) to carry cytotoxic T cell (Tc) epitopes. MPyV VLPs can be assembled in vitro from purified subunits composed of pentamers of the major coat protein VP1 called capsomeres; and this approach provides fine control over VLP composition and structure. Tc-epitopes are often highly hydrophobic, which poses a significant bioengineering challenge and two distinct approaches were pursued to determine the optimal delivery strategy of Influenza Tc-epitopes by MPyV VLP. Firstly, identified epitopes were externally presented using established sites for peptide insertion on the MPyV major coat protein, VP1. Secondly, polypeptides possessing multiple epitopes were encapsidated within VP1 VLP using precise elements of the minor coat protein VP2/3 that interact with the interior cavity of capsomeres. Hydrophobicity-related capsomere aggregation arising from single Tc epitope presentation was successfully circumvented by engineering charged amino acids (DD) flanking the epitope displayed on a surface-exposed loop of VP1 and by use of an optimised buffer condition. A multiparametric, high-throughput buffer screen was implemented to optimise pH and buffer additives during affinity tag removal. Stable modified capsomere recovered from this reaction were assembly competent in the presence of L-Arginine, and VLP yield was high when modular capsomeres were co-assembled with unmodified VP1 capsomeres forming stable mosaic VLPs. The ability of the MPyV VLP to encapsidate foreign protein was first evaluated and optimised with green fluorescent protein (GFP) as a model protein to enable monitoring and analysis.A minimal VP1-interacting sequence was defined from the carboxy-terminus of VP2/3 (VP2C) and fused to the amino-terminus of GFP, ensuring internalisation of the reporter protein. VP1:VP2C-GFP capsomere complexes were successfully recovered when VP1 was co-expressed with VP2C-GFP, whereas complex formation was not observed when VP1 and VP2C-GFP were mixed in vitro.Recovered capsomere complexes were assembly competent and amount of encapsidated GFP was controllable when VP1:VP2C-GFP capsomere complexes were co-assembled with unmodified VP1 capsomeres in a defined molar ratio. The encapsidation approach was then applied to a subdomain of the Influenza matrix protein M1 by co-expressing VP1 with VP2C-M1-I. M1-I capsomere complexes were assembly competent as indicated by gel electrophoresis, dynamic light scattering, and transmission electron microscopy. To enable recovery of VLPs for analysis and characterisation as well as to confirm encapsidation and the formation of mosaic VLPs, a semi-preparative size exclusion chromatography method was developed. This research was able to address bioengineering challenges posed by hydrophobic epitope presentation and developed MPyV

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Downstream Processing

Gottschalk

2012

Pharmaceutical Biotechnology

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Gel Filtration: Size Exclusion Chromatography

Cited by 35 publications

References 164 publications

Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

Downstream Processing

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