Size-exclusion chromatography

Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers. Applications The main a

Preparation and characterization of Poly(Ethylene Glycol)/gamma-CD Polyrotaxanes using beta-CD derivatives as a capping reagent

Blaise Tardy

As living systems understanding got better, as life could be modeled in a batch, using immortalized cells, researches in fundamental sciences found startling applications of advanced techniques on living systems. As the biomaterial field developed various molecules were found to have very interesting applications on living systems. Polyrotaxane is one of these particularly interesting molecules. It is a non covalently bonded molecular necklace produced by the threading of cyclic molecules onto a linear polymer in order to form a pseudopolyrotaxane, the capping of this pseudopolyrotaxane to avoid de-threading of the cyclic compounds forms a polyrotaxane. This compound has already proven its versatility through various application including drug delivery systems, DNA delivery agent, molecular machine, hydrogel building block, resin stabilizer and a lot more. Cyclodextrins (CDs) and Polyester are known to form a pseudopolyrotaxane. CDs are composed of 6 to 8 glucopyranose forming a toroid shaped molecule that can contain different molecules depending on the solvent, experimental conditions and the number of sugar subunits. In this project an attempt has been made to synthesize a special type of polyrotaxane that would allow the whole complex to bare a higher degree of freedom compared to the ones currently documented. In order to do so the widest CD has been used (Gamma-CD, GCD), the chosen host has been poly(ethylene glycol) (PEG) chains of varying molecular weight and the capping reagent used was beta-CD derivatives. Furthermore since current literature hasn't documented it so well and its properties are essential to this study, some more characterization of the PEG/GCD pseudopolyrotaxane has been made. The Macromolecules have been characterized by maldi tof mass spectroscopy(MALDI-T-MS), electro spray ionization (ESI) mass detection, X-ray diffraction(XRD), gel phase chromatography (GPC) and liquid Nuclear magnetic resonance (NMR). The beta CD and poly(ethylene glycol) derivatives have been characterized using Fourier transform infra red spectroscopy (FTIR), NMR, MALDI-Toff MS, ESI and thin layer Chromatography (TLC)

2009

Structural differences of amyloid-beta fibrils revealed by antibodies from phage display

Patrick Droste, Hilal Lashuel

Background: Beside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer's disease (AD) being composed of aggregated fibrils of beta-amyloid (A beta). During the underlying fibrillogenic pathway, starting from a surplus of soluble A beta and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of beta-amyloid and to have tools at hand to monitor the distribution of aggregates in the brain or even act as disease modulators, it is essential to develop highly sensitive antibodies that can discriminate between diverse aggregates of A beta. Results: Here we report the generation and characterization of a variety of amyloid-beta specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from A beta 42 peptides. These antigens were used for the generation of two A beta 42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naive human phage display libraries resulted in multiple unique binders specific for amyloid-beta. Three of the obtained antibodies target the N-terminal part of A beta 42 although with varying epitopes, while another scFv binds to the a-helical central region of the peptide. The affinities of the antibodies to various A beta 42 aggregates as well as their ability to interfere with fibril formation and disaggregation of preformed fibrils were determined. Most significantly, one of the scFv is fibril-specific and can discriminate between two different fibril forms resulting from variations in the acidity of the milieu during fibrillogenesis. Conclusion: We demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti beta-amyloid scFvs that are capable of accurately discriminating between minute conformational differences.

Biomed Central Ltd2015

Preparation and characterization of Poly(Ethylene Glycol)/gamma-CD Polyrotaxanes using beta-CD derivatives as a capping reagent

Blaise Tardy

2009

Structural differences of amyloid-beta fibrils revealed by antibodies from phage display

Patrick Droste, Hilal Lashuel

Biomed Central Ltd2015

Preparation and characterization of Poly(Ethylene Glycol)/gamma-CD Polyrotaxanes using beta-CD derivatives as a capping reagent

Nanoparticle electrostatic exclusion and characterization using fTIRF

Structural differences of amyloid-beta fibrils revealed by antibodies from phage display

Nanoparticle electrostatic exclusion and characterization using fTIRF

Preparation and characterization of Poly(Ethylene Glycol)/gamma-CD Polyrotaxanes using beta-CD derivatives as a capping reagent

Structural differences of amyloid-beta fibrils revealed by antibodies from phage display