Lee J.Y.,Yonsei University |
Choi D.,Yonsei University |
Johan C.,Wyatt Technology Europe GmbH |
Moon M.H.,Yonsei University
Journal of Chromatography A | Year: 2011
In this article, a simple experimental approach to improve lipoprotein separation and detection in flow field-flow fractionation (FlFFF) is detailed. Lipoproteins are globular particles composed of lipids and proteins in blood serum and their roles include transferring fats and cholesterols through blood vessels throughout the body. Especially, presence of small, dense low-density lipoproteins (LDL) is associated with cardiovascular risk. Two experimental approaches were explored in this study: an increase in the reproducibility of LDL particle separation by implementing a guard channel prior to an asymmetrical FlFFF (AFlFFF) channel in order to deplete small molecular weight serum proteins and reducing the required injection volume of a serum sample by implementing fluorescence detection. The guard channel was made of a simple hollow fiber module so that the serum sample can be washed with the help of radial flow prior to injection into the AFlFFF channel. The channel was tested with protein standards and serum samples to ensure precision of the retention time and the protein recovery rate. A fluorescent phospholipid dye was utilized to label lipoprotein particles before separation for fluorescence detection, which resulted in a reduction of the required injection volume of serum. © 2010 Elsevier B.V.
Johann C.,Wyatt Technology Europe GmbH |
Elsenberg S.,Superon GmbH |
Schuch H.,Superon GmbH |
Rosch U.,Superon GmbH
Analytical Chemistry | Year: 2015
A new FFF method is presented which combines asymmetrical flow-FFF (AF4) and electrical FFF (ElFFF) in one channel to electrical asymmetrical flow-FFF (EAF4) to overcome the restrictions of pure ElFFF. It allows for measuring electrophoretic mobility (μ) as a function of size. The method provides an absolute value and does not require calibration. Results of μ for two particle standards are in good agreement with values determined by phase analysis light scattering (PALS). There is no requirement for low ionic strength carriers with EAF4. This overcomes one of the main limitations of ElFFF, making it feasible to measure proteins under physiological conditions. EAF4 has the capability to determine μ for individual populations which are resolved into separate peaks. This is demonstrated for a mixture of three polystyrene latex particles with different sizes as well as for the monomer and dimer of BSA and an antibody. The experimental setup consists of an AF4 channel with added electrodes; one is placed beneath the frit at the bottom wall and the other covers the inside of the upper channel plate. This design minimizes contamination from the electrolysis reactions by keeping the particles distant from the electrodes. In addition the applied voltage range is low (1.5-5 V), which reduces the quantity of gaseous electrolysis products below a threshold that interferes with the laminar flow profile or detector signals. Besides measuring μ, the method can be useful to improve the separation between sample components compared to pure flow-FFF. For two proteins (BSA and a monoclonal antibody), enhanced resolution of the monomer and dimer is achieved by applying an electric field. © 2015 American Chemical Society.
Schleeh T.,Center De Recherche Public Gabriel Lippmann |
Madau M.,Center De Recherche Public Gabriel Lippmann |
Roessner D.,Wyatt Technology Europe GmbH
Carbohydrate Polymers | Year: 2014
The adaptation of alginates to food and pharmaceutical specifications is limited to aqueous chemistry due to the insolubility of sodium alginate (Na-Alg) and the insufficient solubility of tetrabutylammonium alginate (TBA-Alg) in organic solvents. In the present investigation, these restrictions were resolved by optimizing the solubility of TBA-Alg by improving its synthesis from Na-Alg via heterogeneous acidification with hydrochloric and formic acid, followed by neutralization with tetrabutylammonium hydroxide. The best acidification results were achieved with formic acid, because the reaction controlling solubility of the by-product in the acidic solvent was improved in comparison to hydrochloric acid. The solubility of TBA-Alg in polar aprotic organic solvents improved by increasing the degree of TBA substitution (DSTBA), decreasing the molecular weight of TBA-Alg and increasing the relative permittivity of the solvent. The best TBA-Algs, with DSTBA = 0.95 and relative high molecular weights, gave optically clear solutions with a turbidity of about 1 NTU. © 2014 Elsevier Ltd. All rights reserved.
Heim M.,University of Bayreuth |
Ackerschott C.B.,TU Munich |
Ackerschott C.B.,Wyatt Technology Europe GmbH |
Scheibel T.,University of Bayreuth
Journal of Structural Biology | Year: 2010
The capture spiral of a spider's orb web is made of flagelliform silk, providing high elasticity and an outstanding toughness, perfectly suited for trapping prey. Flagelliform silk comprises mainly one single protein (FLAG) with an estimated molecular weight of 360. kDa. We engineered constructs mimicking distinct domains of FLAG (eFLAG) and produced them recombinantly to analyze the structure-function relationship of FLAG domains and assembly properties of FLAG. While in solution the small carboxy-terminal domain is structured, domains from the repetitive core region adopt a conformation typical for intrinsically unstructured proteins. To investigate the influence of the respective domains on solubility and assembly, we tested the aggregation behaviour of individual domains and domain ensembles in presence of conditions known to trigger silk assembly. Both, the length of the repetitive core domain as well as the presence of the carboxy-terminal non-repetitive domain showed impact on eFLAG aggregation. © 2009 Elsevier Inc.
Kostogrys R.B.,Jagiellonian University |
Johann C.,Wyatt Technology Europe GmbH |
Czyzynska I.,Agricultural University of Krakow |
Franczyk-Zarow M.,Agricultural University of Krakow |
And 12 more authors.
Journal of Nutrition, Health and Aging | Year: 2015
Introduction: Low Carbohydrate High Protein diet represents a popular strategy to achieve weight loss. Objective: The aim of this study was to characterize effects of low carbohydrate, high protein diet (LCHP) on atherosclerotic plaque development in brachiocephalic artery (BCA) in apoE/LDLR−/− mice and to elucidate mechanisms of proatherogenic effects of LCHP diet. Materials and Methods: Atherosclerosis plaques in brachiocephalic artery (BCA) as well as in aortic roots, lipoprotein profile, inflammation biomarkers, expression of SREBP-1 in the liver as well as mortality were analyzed in Control diet (AIN-93G) or LCHP (Low Carbohydrate High Protein) diet fed mice. Results: Area of atherosclerotic plaques in aortic roots or BCA from LCHP diet fed mice was substantially increased as compared to mice fed control diet and was characterized by increased lipids and cholesterol contents (ORO staining, FT-IR analysis), increased macrophage infiltration (MOMA-2) and activity of MMPs (zymography). Pro-atherogenic phenotype of LCHP fed apoE/LDLR−/− mice was associated with increased plasma total cholesterol concentration, and in LDL and VLDL fractions, increased TG contents in VLDL, and a modest increase in plasma urea. LCHP diet increased SCD-1 index, activated SREBP-1 transcription factor in the liver and triggered acute phase response as evidence by an increased plasma concentration of haptoglobin, CRP or AGP. Finally, in long-term experiment survival of apoE/LDLR−/− mice fed LCHP diet was substantially reduced as compared to their counterparts fed control diet suggesting overall detrimental effects of LCHP diet on health. Conclusions: The pro-atherogenic effect of LCHP diet in apoE/LDLR−/− mice is associated with profound increase in LDL and VLDL cholesterol, VLDL triglicerides, liver SREBP-1 upregulation, and systemic inflammation. © 2015, Serdi and Springer-Verlag France.