Browse
Submit Data
Databases
API
Help

Dataset Information

31 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A method for rapid high-throughput biophysical analysis of proteins.

ABSTRACT: Quantitative determination of protein thermodynamic stability is fundamental to many research areas, both basic and applied. Although chemical-induced denaturation is the gold-standard method, it has been replaced in many settings by semi-quantitative approaches such as thermal stability measurements. The reason for this shift is that chemical denaturation experiments are labour-intensive, sample-costly and time-consuming, and it has been assumed that miniaturisation to a high-throughput format would not be possible without concomitantly comprising data quality. Here we exploit current technologies to create a high-throughput label-free chemical denaturation method that is capable of generating replicate datasets on multiple proteins in parallel on a timescale that is at least ten times faster, much more economical on sample, and with the potential for superior data quality, than the conventional methods used in most research labs currently.

SUBMITTER: Perez-Riba A

PROVIDER: S-EPMC5567296 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A method for rapid high-throughput biophysical analysis of proteins.

Perez-Riba Albert A Itzhaki Laura S LS

Scientific reports 20170822 1

Quantitative determination of protein thermodynamic stability is fundamental to many research areas, both basic and applied. Although chemical-induced denaturation is the gold-standard method, it has been replaced in many settings by semi-quantitative approaches such as thermal stability measurements. The reason for this shift is that chemical denaturation experiments are labour-intensive, sample-costly and time-consuming, and it has been assumed that miniaturisation to a high-throughput format ...[more]

PMID: 28831058

Similar Datasets

A rapid, high-throughput method for determining chronological lifespan in budding yeast.

Project description:The budding yeast Saccharomyces cerevisiae is a major model system in the study of aging. Like metazoans, yeast lifespan is extended by caloric restriction and treatment with pharmacological agents which extend lifespan. A major workhorse of aging research in budding yeast is the chronological lifespan assay. Traditionally, chronological lifespan assays consist of taking regular samples of aging yeast cultures, plating out aliquots on agar, and counting the resulting colonies. This method, while highly reliable, is labor-intensive and expensive in terms of materials consumed. Here, we report a novel MTT-based method for assessing chronological lifespan in yeast. We show that this method is equal to the colony counting method in its rigorous and reliable measurement of lifespan extension in yeast as a result of caloric restriction, and is able to distinguish known long-lived and short-lived yeast strains. We have further developed this method into a high-throughput assay that allows rapid screening of potential anti-aging compounds as well as yeast strains with altered lifespan. Application of this method permits the rapid identification of anti-aging activities in yeast and may facilitate identification of materials with therapeutic potential for higher animals and, most importantly, humans.

| S-EPMC6706159 | biostudies-literature

A Rapid and High Throughput MIC Determination Method to Screen Uranium Resistant Microorganisms.

Project description:The assessment of minimum inhibitory concentration (MIC) is a conventional technique used for the screening of microbial resistance against antibiotics, biocides, and contaminants such as heavy metals. However, as part of our ongoing work, we have observed biases associated with using traditional liquid MIC method to screen microbial heavy metal resistance, including both bacterial and fungal strains. Specifically, the addition of uranium into synthetic media causes immediate precipitation prior to the initiation of microbial growth, thus hampering the optical density measurements, and the obtained MIC values are thus flawed and inaccurate. To address this discrepancy, we report the optimization and development of a serial-dilution-based MIC method conducted on solid growth media supplemented with uranium, which is more accurate, relative to the testing of MICs performed in liquid cultures. Notably, we report on the efficacy of this method to screen not only bacteria that are resistant to uranium but also demonstrate the successful application to yeast and fungal isolates, for their ability to resist uranium, is more accurate and sensitive relative to the liquid method. We believe that this newly developed method to screen heavy metal resistance, such as uranium, is far superior to the existing liquid MIC method and propose replacing the liquid assay with the solid plate MIC reported herein.

| S-EPMC7189662 | biostudies-literature

A method for high-throughput gene expression signature analysis.

Project description:Genome-wide transcriptional profiling has shown that different biologic states (for instance, disease and response to pharmacologic manipulation) can be recognized by the expression pattern of relatively small numbers of genes. However, the lack of a practical and cost-effective technology for detection of these gene expression 'signatures' in large numbers of samples has severely limited their exploitation in important medical and pharmaceutical discovery applications. Here, we describe a solution based on the combination of ligation-mediated amplification with an optically addressed microsphere and flow cytometric detection system.

| S-EPMC1779561 | biostudies-literature

The rapid "teabag" method for high-end purification of membrane proteins.

Project description:Overproduction and purification of membrane proteins are generally challenging and time-consuming procedures due to low expression levels, misfolding, and low stability once extracted from the membrane. Reducing processing steps and shortening the timespan for purification represent attractive approaches to overcome some of these challenges. We have therefore compared a fast "teabag" purification method with conventional purification for five different membrane proteins (MraY, AQP10, ClC-1, PAR2 and KCC2). Notably, this new approach reduces the purification time significantly, and the quality of the purified membrane proteins is equal to or exceeds conventional methods as assessed by size exclusion chromatography, SDS-PAGE and downstream applications such as ITC, crystallization and cryo-EM. Furthermore, the method is scalable, applicable to a range of affinity resins and allows for parallelization. Consequently, the technique has the potential to substantially simplify purification efforts of membrane proteins in basic and applied sciences.

| S-EPMC7528119 | biostudies-literature

Bazam: a rapid method for read extraction and realignment of high-throughput sequencing data.

Project description:The vast quantities of short-read sequencing data being generated are often exchanged and stored as aligned reads. However, aligned data becomes outdated as new reference genomes and alignment methods become available. Here we describe Bazam, a tool that efficiently extracts the original paired FASTQ from alignment files (BAM or CRAM format) in a format that directly allows efficient realignment. Bazam facilitates up to a 90% reduction in the time for realignment compared to standard methods. Bazam can support selective extraction of read pairs from focused genomic regions for applications such as targeted region analyses, quality control, structural variant calling, and alignment comparisons.

| S-EPMC6472072 | biostudies-literature

Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea.

Project description:Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure-function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.

| S-EPMC7217052 | biostudies-literature

Measuring Protein Aggregation and Stability Using High-Throughput Biophysical Approaches.

Project description:Structure-function relationships of biological macromolecules, in particular proteins, provide crucial insights for fundamental biochemistry, medical research and early drug discovery. However, production of recombinant proteins, either for structure determination, functional studies, or to be used as biopharmaceutical products, is often hampered by their instability and propensity to aggregate in solution in vitro. Protein samples of poor quality are often associated with reduced reproducibility as well as high research and production expenses. Several biophysical methods are available for measuring protein aggregation and stability. Yet, discovering and developing means to improve protein behaviour and structure-function integrity remains a demanding task. Here, we discuss workflows that are made possible by adapting established biophysical methods to high-throughput screening approaches. Rapid identification and optimisation of conditions that promote protein stability and reduce aggregation will support researchers and industry to maximise sample quality, stability and reproducibility, thereby reducing research and development time and costs.

| S-EPMC9149252 | biostudies-literature

A simple, low-cost staining method for rapid-throughput analysis of tumor spheroids.

Project description:Tumor spheroids are becoming an important tool for the investigation of cancer stem cell (CSC) function in tumors; thus, low-cost and high-throughput methods for drug screening of tumor spheroids are needed. Using neurospheres as non-adherent three-dimensional (3-D) cultures, we developed a simple, low-cost acridine orange (AO)-based method that allows for rapid analysis of live neurospheres by fluorescence microscopy in a 96-well format. This assay measures the cross-section area of a spheroid, which corresponds to cell viability. Our novel method allows rapid screening of a panel of anti-proliferative drugs to assess inhibitory effects on the growth of cancer stem cells in 3-D cultures.

| S-EPMC4772731 | biostudies-literature

A rapid, high-throughput, and sensitive PEG-precipitation method for SARS-CoV-2 wastewater surveillance.

Project description:The effective application of wastewater surveillance is dependent on testing capacity and sensitivity to obtain high spatial resolution testing results for a timely targeted public health response. To achieve this purpose, the development of rapid, high-throughput, and sensitive virus concentration methods is urgently needed. Various protocols have been developed and implemented in wastewater surveillance networks so far, however, most of them lack the ability to scale up testing capacity or cannot achieve sufficient sensitivity for detecting SARS-CoV-2 RNA at low prevalence. In the present study, using positive raw wastewater in Hong Kong, a PEG precipitation-based three-step centrifugation method was developed, including low-speed centrifugation for large particles removal and the recovery of viral nucleic acid, and medium-speed centrifugation for the concentration of viral nucleic acid. This method could process over 100 samples by two persons per day to reach the process limit of detection (PLoD) of 3286 copies/L wastewater. Additionally, it was found that the testing capacity could be further increased by decreasing incubation and centrifugation time without significantly influencing the method sensitivity. The entire procedure uses ubiquitous reagents and instruments found in most laboratories to obtain robust testing results. This high-throughput, cost-effective, and sensitive tool will promote the establishment of nearly real-time wastewater surveillance networks for valuable public health information.

| S-EPMC9803703 | biostudies-literature

Rapid and High Throughput Hydroponics Phenotyping Method for Evaluating Chickpea Resistance to Phytophthora Root Rot.

Project description:Phytophthora root rot (PRR) is a major constraint to chickpea production in Australia. Management options for controlling the disease are limited to crop rotation and avoiding high risk paddocks for planting. Current Australian cultivars have partial PRR resistance, and new sources of resistance are needed to breed cultivars with improved resistance. Field- and glasshouse-based PRR resistance phenotyping methods are labour intensive, time consuming, and provide seasonally variable results; hence, these methods limit breeding programs' abilities to screen large numbers of genotypes. In this study, we developed a new space saving (400 plants/m2), rapid (<12 days), and simplified hydroponics-based PRR phenotyping method, which eliminated seedling transplant requirements following germination and preparation of zoospore inoculum. The method also provided post-phenotyping propagation all the way through to seed production for selected high-resistance lines. A test of 11 diverse chickpea genotypes provided both qualitative (PRR symptoms) and quantitative (amount of pathogen DNA in roots) results demonstrating that the method successfully differentiated between genotypes with differing PRR resistance. Furthermore, PRR resistance hydroponic assessment results for 180 recombinant inbred lines (RILs) were correlated strongly with the field-based phenotyping, indicating the field phenotype relevance of this method. Finally, post-phenotyping high-resistance genotypes were selected. These were successfully transplanted and propagated all the way through to seed production; this demonstrated the utility of the rapid hydroponics method (RHM) for selection of individuals from segregating populations. The RHM will facilitate the rapid identification and propagation of new PRR resistance sources, especially in large breeding populations at early evaluation stages.

| S-EPMC10708383 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data