scholarly journals Quality Control Of FITC-labeled Proteins for Interactomics Investigations Using SDS Capillary Gel Electrophoresis and SPR Biosensor

2019 ◽  
Vol 2 (4) ◽  
pp. e00112
Author(s):  
P.V. Ershov ◽  
L.A. Kaluzhskiy ◽  
E.O. Yablokov ◽  
A.S. Ivanov
Keyword(s):  
Gel Electrophoresis ◽  
Binding Capacity ◽  
Fluorescein Isothiocyanate ◽  
Target Protein ◽  
Step Method ◽  
Protein Preparation ◽  
Capillary Gel Electrophoresis ◽  
Protein Protein Interaction ◽  
Spr Biosensor

The technology of dye-labeled proteins has many fields of application, especially in interactomics. The aim of this work was to adapt protocol of conjugation of low molecular weight (12 – 15 kDа) heme-containing proteins with fluorescein isothiocyanate, isomer I, (FITC) for subsequent protein-protein interaction studies. We have monitored the quality of FITC-labeling of the target protein and comparative assessment of its binding capacity. Using the cytochrome C (Mw 12 kDа) as an example, it has been shown that using the three step method approach including conventional spectrophotometry, capillary gel electrophoresis and SPR analysis it is possible to assess: (i) the capability of the FITC-labeled target protein to interact with its protein partner and protein material from tissue lysates, (ii) the fact of dye conjugation with the protein, and (iii) the quality of purification for final protein preparation from unreacted free dye molecules

scholarly journals Annotating proteins with generalized functional linkages

2008 ◽  
Vol 105 (46) ◽  
pp. 17700-17705 ◽  
Author(s):  
Richard Llewellyn ◽  
David S. Eisenberg
Keyword(s):  
Protein Function ◽  
Biological Process ◽  
Computational Method ◽  
Majority Voting ◽  
Target Protein ◽  
Biological Processes ◽  
Gene Products ◽  
Protein Protein Interaction ◽  
Protein Protein Interaction Networks

As genome sequencing outstrips the rate of high-quality, low-throughput biochemical and genetic experimentation, accurate annotation of protein function becomes a bottleneck in the progress of the biomolecular sciences. Most gene products are now annotated by homology, in which an experimentally determined function is applied to a similar sequence. This procedure becomes error-prone between more divergent sequences and can contaminate biomolecular databases. Here, we propose a computational method of assignment of function, termed Generalized Functional Linkages (GFL), that combines nonhomology-based methods with other types of data. Functional linkages describe pairwise relationships between proteins that work together to perform a biological task. GFL provides a Bayesian framework that improves annotation by arbitrating a competition among biological process annotations to best describe the target protein. GFL addresses the unequal strengths of functional linkages among proteins, the quality of existing annotations, and the similarity among them while incorporating available knowledge about the cellular location or individual molecular function of the target protein. We demonstrate GFL with functional linkages defined by an algorithm known as zorch that quantifies connectivity in protein–protein interaction networks. Even when using proteins linked only by indirect or high-throughput interactions, GFL predicts the biological processes of many proteins in Saccharomyces cerevisiae, improving the accuracy of annotation by 20% over majority voting.

Evaluation of the mixing quality of high-viscosity yield stress fluids in a tubular reactor

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Le Xie ◽  
Guangwen He ◽  
Bin Yu ◽  
Shaowei Yan
Keyword(s):  
Yield Stress ◽  
Large Scale ◽  
Molecular Diffusion ◽  
Time History ◽  
High Viscosity ◽  
Cfd Model ◽  
Step Method ◽  
Mean Velocity ◽  
Mixing Quality

Abstract In this study, the mixing quality of high-viscosity yield stress fluid (Carbopol aqueous solution) under laminar and turbulent flow regimes was evaluated through a numerical experimental study. A three-dimensional computational fluid dynamics large-eddy simulation (CFD-LES) model was employed to capture large-scale vortex structures. The proposed CFD model was validated by the experimental data in terms of mean velocity profiles and velocity-time history. Thereafter, the CFD model was applied to simulate the residence time distribution using the tracking technique: tracer pulse method and step method. In addition, the non-ideal flow phenomena caused by molecular diffusion and eddy diffusion were evaluated. The effects of the rheological properties on the mixing performance were also investigated. The presented results can provide useful guidance to enhance mass transfer in reactors with high-viscosity fluids.

scholarly journals CATH functional families predict functional sites in proteins

2020 ◽  
Author(s):  
Sayoni Das ◽  
Harry M Scholes ◽  
Neeladri Sen ◽  
Christine Orengo
Keyword(s):  
Functional Characterization ◽  
Functional Site ◽  
Training Data ◽  
Supplementary Information ◽  
Conserved Residues ◽  
Functional Sites ◽  
Protein Protein Interaction ◽  
Evolutionary Features ◽  
Functional Families

Abstract Motivation Identification of functional sites in proteins is essential for functional characterization, variant interpretation and drug design. Several methods are available for predicting either a generic functional site, or specific types of functional site. Here, we present FunSite, a machine learning predictor that identifies catalytic, ligand-binding and protein–protein interaction functional sites using features derived from protein sequence and structure, and evolutionary data from CATH functional families (FunFams). Results FunSite’s prediction performance was rigorously benchmarked using cross-validation and a holdout dataset. FunSite outperformed other publicly available functional site prediction methods. We show that conserved residues in FunFams are enriched in functional sites. We found FunSite’s performance depends greatly on the quality of functional site annotations and the information content of FunFams in the training data. Finally, we analyze which structural and evolutionary features are most predictive for functional sites. Availabilityand implementation https://github.com/UCL/cath-funsite-predictor. Contact [email protected] or [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Rapid capillary gel electrophoresis analysis of human milk oligosaccharides for food additive manufacturing in-process control

2021 ◽  
Author(s):  
Marton Szigeti ◽  
Agnes Meszaros-Matwiejuk ◽  
Dora Molnar-Gabor ◽  
Andras Guttman
Keyword(s):  
Additive Manufacturing ◽  
Process Control ◽  
Human Milk ◽  
Gel Electrophoresis ◽  
Process Analysis ◽  
Food Additive ◽  
Human Milk Oligosaccharides ◽  
Milk Oligosaccharides ◽  
Capillary Gel Electrophoresis

AbstractIndustrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2′-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2′- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control. Graphical abstract

Optimized Large Scale Synthesis of Phosphorothioate Oligonucleotides on PS-PEG Supports

1997 ◽  
Vol 52 (1) ◽  
pp. 110-116
Author(s):  
Michael Gerster ◽  
Martin Maier ◽  
Nils Clausen ◽  
Jens Schewitz ◽  
Ernst Bayer
Keyword(s):  
Gel Electrophoresis ◽  
Continuous Flow ◽  
Large Scale ◽  
Electrospray Mass Spectrometry ◽  
Small Scale ◽  
Batch Mode ◽  
Crucial Step ◽  
Capillary Gel Electrophoresis ◽  
Phosphoramidite Chemistry ◽  
Large Scale Synthesis

Sulphurization is a crucial step during synthesis of phosphorothioate oligonucleotides. Insufficient reaction leads to inhomogeneous products with phosphodiester defects and subsequently to destabilization of the oligomers in biological media. To achieve a maximum extent of sulphur incorporation, various sulphurizing agents have been investigated. Solely, the use of Beaucage reagent provided satisfactory results on PS-PEG supports. Based on our investigations in small scale synthesis (1 μmol) with continuous-flow technique, upscaling to the 0.1-0.25 mmolar range has been achieved using a peptide synthesizer. The syntheses were performed in batch mode with standard phosphoramidite chemistry. Additionally, large scale synthesis of a phosphodiester oligonucleotide has been carried out on PS-PEG with optimized protocols and compared to small scale synthesis on different supports. Products were analysed by 31P NMR, capillary gel electrophoresis and electrospray mass spectrometry. An extent of sulphurization of 99% and coupling effiencies of more than 99% were obtained and the products proved to have similar purity compared to small scale syntheses on CPG

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