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Journal of Biomedicine & Biotechnology 2009Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent... (Review)
Review
Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.
Topics: Chemical Fractionation; Chromatography; DNA; Proteins; RNA
PubMed: 20011662
DOI: 10.1155/2009/574398 -
Journal of Molecular Biology Nov 2018The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In...
The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.
Topics: Animals; Cell Nucleus; Chemical Fractionation; Cytoplasm; Guidelines as Topic; In Vitro Techniques; Liquid-Liquid Extraction; Peptide Termination Factors; Phase Transition; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Solid Phase Extraction
PubMed: 29944854
DOI: 10.1016/j.jmb.2018.06.038 -
Nature Methods Feb 2008In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we... (Review)
Review
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
Topics: Chemical Fractionation; Chemistry, Physical; Protein Engineering; Proteomics; Recombinant Proteins
PubMed: 18235434
DOI: 10.1038/nmeth.f.202 -
Biosensors & Bioelectronics Dec 2020Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease... (Review)
Review
Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.
Topics: Betacoronavirus; COVID-19; Chemical Fractionation; Communicable Diseases; Coronavirus Infections; Equipment Design; Humans; Lab-On-A-Chip Devices; Nucleic Acid Amplification Techniques; Nucleic Acids; Pandemics; Plant Diseases; Pneumonia, Viral; Point-of-Care Systems; SARS-CoV-2
PubMed: 32942143
DOI: 10.1016/j.bios.2020.112592 -
Scientific Reports Mar 2019Exosomes have received significant attention for their role in pathobiological processes and are being explored as a tool for disease diagnosis and management....
Exosomes have received significant attention for their role in pathobiological processes and are being explored as a tool for disease diagnosis and management. Consequently, various isolation methods based on different principles have been developed for exosome isolation. Here we compared the efficacy of four kits from Invitrogen, 101Bio, Wako and iZON along with conventional ultracentrifugation-based method for exosome yield, purity and quality. Cell culture supernatant was used as an abundant source of exosomes, and exosome quantity, size-distribution, zeta-potential, marker-expression and RNA/protein quality were determined. The Invitrogen kit gave the highest yield but the preparation showed broader size-distribution likely due to microvesicle co-precipitation and had the least dispersion stability. Other preparations showed <150 nm size range and good stability. Preparation from iZON column; however, had a broader size-distribution in the lower size range suggestive of some impurities of non-vesicular aggregates. RNA quality from all preparations was comparable; however, proteins from Invitrogen method-based exosomal preparation showed polyethylene glycol (PEG) contamination in mass spectrometry. Chemical impurities from the precipitant could also be the cause of toxicity of Invitrogen method-based exosomal preparation in biological growth measurement assay. Together, these findings should serve as a guide to choose and further optimize exosome isolation methods for their desired downstream applications.
Topics: Chemical Fractionation; Culture Media, Conditioned; Exosomes; Humans; Mass Spectrometry; Particle Size; Workflow
PubMed: 30926864
DOI: 10.1038/s41598-019-41800-2 -
Bioanalysis May 2016
Topics: Animals; Chemical Fractionation; Chromatography; Dried Blood Spot Testing; Drug Discovery; Humans; Lab-On-A-Chip Devices; Mass Spectrometry; Microfluidic Analytical Techniques; Organ Culture Techniques
PubMed: 27109573
DOI: 10.4155/bio-2016-0081 -
Molecules (Basel, Switzerland) Nov 2022Xanthanolides were particularly characteristic of the genus , which exhibited broad biological effects and have drawn much attention in pharmacological application. The... (Review)
Review
Xanthanolides were particularly characteristic of the genus , which exhibited broad biological effects and have drawn much attention in pharmacological application. The review surveyed the structures and bioactivities of the xanthanolides in the genus , and summarized the synthesis tactics of xanthanolides. The results indicated that over 30 naturally occurring xanthanolides have been isolated from the genus in monomeric, dimeric and trimeric forms. The bioassay-guided fractionation studies suggested that the effective fractions on antitumor activities were mostly from weak polar solvents, and xanthatin () was the most effective and well-studied xanthanolide. The varieties of structures and structure-activity relationships of the xanthanolides had provided the promising skeleton for the further study. The review aimed at providing guidance for the efficient preparation and the potential prospects of the xanthanolides in the medicinal industry.
Topics: Xanthium; Chemical Fractionation
PubMed: 36500229
DOI: 10.3390/molecules27238136 -
Journal of Chromatography. A Dec 2023Field-flow fractionation (FFF) with its several variants, has developed into a mature methodology. The scope of the FFF investigations has expanded, covering both a wide... (Review)
Review
Field-flow fractionation (FFF) with its several variants, has developed into a mature methodology. The scope of the FFF investigations has expanded, covering both a wide range of basic studies and especially a wide range of analytical applications. Special attention of this review is given to the achievements of FFF with reference to recent applications in the fractionation, isolation, and purification of biomacromolecules, and from which especially those of (in alphabetical order) bacteria, cells, extracellular vesicles, liposomes, lipoproteins, nucleic acids, and viruses and virus-like particles. In evaluating the major approaches and trends demonstrated since 2012, the most significant biomacromolecule applications are compiled in tables. It is also evident that asymmetrical flow field-flow fractionation is by far the most dominant technique in the studies. The industry has also shown current interest in FFF and adopted it in some sophisticated fields. FFF, in combination with appropriate detectors, handles biomacromolecules in open channel in a gentle way due to the lack of shear forces and unwanted interactions caused by the stationary phase present in chromatography. In addition, in isolation and purification of biomacromolecules quite high yields can be achieved under optimal conditions.
Topics: Chemical Fractionation; Fractionation, Field Flow; Lipoproteins; Chromatography; Liposomes
PubMed: 37944435
DOI: 10.1016/j.chroma.2023.464492 -
Bioresource Technology Feb 2023Lignocellulosic biomass is one of the most important renewable materials to replace carbon-based fossil resources. Solvent-based fractionation is a promising route for... (Review)
Review
Lignocellulosic biomass is one of the most important renewable materials to replace carbon-based fossil resources. Solvent-based fractionation is a promising route for fractionation of biomass into its major components. Processing is governed by the employed solvent-systems properties. This review sheds light on the factors governing both dissolution and potential reactivities of the chemical structures present in lignocellulose, highlighting how proper understanding of the underlying mechanisms and interactions between solute and solvent help to choose proper systems for specific fractionation needs. Structural and chemical differences between the carbohydrate-based structural polymers and lignin require very different solvents capabilities in terms of causing and eventually stabilizing conformational changes and consequent activation of bonds to be cleaved by other active components in the. A consideration of potential depolymerization events during dissolution and energetic aspects of the dissolution process considering the contribution of polymer functionalities allow for a mapping of solvent suitability for biomass fractionation.
Topics: Solvents; Biomass; Lignin; Carbohydrates; Chemical Fractionation
PubMed: 36496118
DOI: 10.1016/j.biortech.2022.128447 -
Biomedical Chromatography : BMC May 2016Lipids make up a diverse subset of biomolecules that are responsible for mediating a variety of structural and functional properties as well as modulating cellular... (Review)
Review
Lipids make up a diverse subset of biomolecules that are responsible for mediating a variety of structural and functional properties as well as modulating cellular functions such as trafficking, regulation of membrane proteins and subcellular compartmentalization. In particular, phospholipids are the main constituents of biological membranes and play major roles in cellular processes like transmembrane signaling and structural dynamics. The chemical and structural variety of lipids makes analysis using a single experimental approach quite challenging. Research in the field relies on the use of multiple techniques to detect and quantify components of cellular lipidomes as well as determine structural features and cellular organization. Understanding these features can allow researchers to elucidate the biochemical mechanisms by which lipid-lipid and/or lipid-protein interactions take place within the conditions of study. Herein, we provide an overview of essential methods for the examination of lipids, including extraction methods, chromatographic techniques and approaches for mass spectrometric analysis.
Topics: Chemical Fractionation; Chromatography; Lipids; Mass Spectrometry
PubMed: 26762903
DOI: 10.1002/bmc.3683