Use Of Gel Electrophoresis Research Articles

Screening Carbon Nano Materials for Preventing Amyloid Protein Aggregation by Adopting a Facile Method.

The soluble-to-toxic transformation of intrinsically disordered amyloidogenic proteins such as amyloid beta (Aβ), α-synuclein, mutant Huntingtin Protein (mHTT) and islet amyloid polypeptide (IAPP) among others are associated with disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and Type 2 Diabetes (T2D), respectively. The dissolution of mature fibrils and toxic amyloidogenic intermediates, including oligomers, continues to be the pinnacle in the treatment of neurodegenerative disorders. Yet, methods to effectively and quantitatively report on the interconversion between amyloid monomers, oligomers and mature fibrils fall short. Here we describe a simplified method that implements the use of gel electrophoresis to address the transformation between soluble monomeric amyloid proteins and mature amyloid fibrils. The technique implements an optimized but well-known, simple, inexpensive, and quantitative assessment previously used to assess the oligomerization of amyloid monomers and subsequent amyloid fibrils. This method facilitates the screening of small molecules that disintegrate oligomers and fibrils into monomers, dimers, and trimers and/or retain amyloid proteins in their monomeric forms. Most importantly, our optimized method diminishes existing barriers associated with existing (alternative) techniques to evaluate fibril formation and intervention.

Physicochemical Characterization of Cassava (Manihot esculenta) and Potato (Ipomea batatas) Varieties from Southeast of Nigeria in Comparison with Agarose for use in the Separation of Deoxyribonucleic Acid

Starch is an essential biomaterial and food product globally used for different purposes. This study determined the physicochemical composition of starch extracted from selected cassava (M.ExOboma I and M.ExOboma II) and sweet potato (I.ExOboma I and I.ExOboma II) varieties in comparison with agarose for use in the separation of deoxyribonucleic acid. Starch extraction was carried out using standard procedure. Granule morphology and amylose-amylopectin contents were determined using Polarized Light Microscopy and Dual Wavelength Iodine-Binding methods respectively. Relationships between starch properties were determined using correlation analysis. The result of the starch granule morphology revealed that there was no significant difference (p > 0.05) in the pore sizes of M.ExOboma I (5.527 ± 1.9346 µm) and M.ExOboma II (5.650 ± 1.0472 µm). Similarly, I.ExOboma I (8.275 ± 1.5064 µm) and I.ExOboma II (6.075 ± 2.3838 µm) showed no significant difference (p > 0.05) in their pore sizes. Meanwhile, the pore size of agarose was reported to be within the range of 6 to 35μm. No significant difference (p > 0.05) was also observed in the ratios of amylose and amylopectin (Am:Ap) contents between the varieties of cassava (M.ExOboma I 25:75 and M.ExOboma II 24:77) and sweet potato (I.ExOboma I 26:72 and I.ExOboma II 27:75). No significant difference (P > 0.05) between the pH range values (7.7 to 7.8) of cassava and sweet potato with that of agarose (7.5). The study also revealed that there were significant differences (p ≤ 0.05) in the clarity as well as the purity level of the starch samples compared to agarose. The results of this study are of great significance in predicting starch functionality and will form a basis for the improvement of the physicochemical attributes of starch for use in gel electrophoresis for the separation of biomolecules.

Approaches for the sensitive detection of rare base and prime editing events.

Precision chemistry entailing user-directed nucleotide substitutions and template-specified repair can be facilitated by base editing and prime editing, respectively. Recently, the diversification of adenine, cytosine, and prime editor variants obliges a considered, high-throughput evaluation of these tools for optimized, end-point applications. Herein, we outline novel, cost-effective and scalable approaches for the rapid detection of base editing and prime editing outcomes using gel electrophoresis. For base editing, we exploit primer mismatch amplification (SNP genotyping) for the gel-based detection of base editing efficiencies as low as 0.1%. For prime editing, we describe a one-pot reaction combining polymerase chain reaction (PCR) amplification of the target region with restriction digestion (restriction fragment length polymorphism; RFLP). RFLP enables the rapid detection of insertion or deletion events in under 2.5h from genomic DNA extraction. We show that our method of SNP genotyping is amenable to both endogenous target loci as well as transfected, episomal plasmid targets in BHK-21 cells. Next, we validate the incidence of base and prime editing by describing Sanger sequencing and next-generation sequencing (NGS) workflows for the accurate validation and quantification of on-target editing efficiencies. Our workflow details three different methods for the detection of rare base and prime editing events, enabling a tiered approach from low to high resolution that makes use of gel electrophoresis, Sanger sequencing, and NGS.

Assessing and Improving Protein Sample Quality.

One essential prerequisite of any experiment involving a purified protein, such as interaction studies or structural and biophysical characterization, is to work with a "good-quality" sample in order to ensure reproducibility and reliability of the data. Here, we define a "good-quality" sample as a protein preparation that fulfills three criteria: (1) the preparation contains a protein that is pure and soluble and exhibits structural and functional integrity, (2) the protein must be structurally homogeneous, and (3) the preparation must be reproducible. To ensure effective quality control (QC) of all these parameters, we suggest tofollow a simple workflow involving the use of gel electrophoresis, light scattering, and spectroscopic experiments. We describe the techniques used in every step of this workflow and provide easy-to-use standard protocols for each step.

Nuclease Degradation Analysis of DNA Nanostructures Using Gel Electrophoresis.

Custom-built DNA nanostructures are now used in applications such as biosensing, molecular computation, biomolecular analysis, and drug delivery. While the functionality and biocompatibility of DNA makes DNA nanostructures useful in such applications, the field faces a challenge in making biostable DNA nanostructures. Being a natural material, DNA is most suited for biological applications, but is also easily degraded by nucleases. Several methods have been employed to study the nuclease degradation rates and enhancement of nuclease resistance. This protocol describes the use of gel electrophoresis to analyze the extent of nuclease degradation of DNA nanostructures and to report degradation times, kinetics of nuclease digestion, and evaluation of biostability enhancement factors. © 2020 Wiley Periodicals LLC. Basic Protocol: Timed analysis of nuclease degradation of DNA nanostructures Support Protocol: Calculating biostability enhancement factors.

Assessment of apoptotic impact of ICD-85 in A549 cancer cells & HEK293 normal cells

Objective(s)One of the sources of medically useful drugs is animal venom. The significance of venom regarding cancer treatment has become more apparent. ICD-85 active fraction is an integration of three peptides, extracted from the venom of two independent species of venomous animals and exhibits anticancer properties. This paper assesses comparative ICD-85 activity in HEK293 normal cells and A549 cancer cells. Materials and methodsCytotoxicity was evaluated by Cell morphology analysis, MTTassay and Neutral Red Uptake (NRU). Apoptosis in A549 and HEK293 cells were investigated by assessing the attributes pattern of DNA laddering formed in the apoptotic cells by the use of gel electrophoresis. The caspase-8 colorimetric assay kit was used to assay caspase-8 activity. ResultsThe results showed that ICD-85 significantly inhibited A549 cells growth with IC50 value of 22.23 ± 2.42 μg/ml by MTT assay through induction of apoptosis. Further studies showed that ICD-85 activated death receptor pathway and the activation of caspase-8, resulting in A549 cell apoptosis. However, caspase-8 activity did not show considerable changes in HEK293 normal cells. ConclusionTaken together, this study demonstrated that ICD-85 inhibited A549 the growth of cancel cells via apoptosis induction.

Detection of Macromolecular Fractions in HCN Polymers Using Electrophoretic and Ultrafiltration Techniques.

Elucidating the origin of life involves synthetic as well as analytical challenges. Herein, for the first time, we describe the use of gel electrophoresis and ultrafiltration to fractionate HCN polymers. Since the first prebiotic synthesis of adenine by Oró, HCN polymers have gained much interest in studies on the origins of life due to the identification of biomonomers and related compounds within them. Here, we demonstrate that macromolecular fractions with electrophoretic mobility can also be detected within HCN polymers. The migration of polymers under the influence of an electric field depends not only on their sizes (one-dimensional electrophoresis) but also their different isoelectric points (two-dimensional electrophoresis, 2-DE). The same behaviour was observed for several macromolecular fractions detected in HCN polymers. Macromolecular fractions with apparent molecular weights as high as 250 kDa were detected by tricine-SDS gel electrophoresis. Cationic macromolecular fractions with apparent molecular weights as high as 140 kDa were also detected by 2-DE. The HCN polymers synthesized were fractionated by ultrafiltration. As a result, the molecular weight distributions of the macromolecular fractions detected in the HCN polymers directly depended on the synthetic conditions used to produce these polymers. The implications of these results for prebiotic chemistry will be discussed.

Condensation of plasmid DNA by benzyl hydraphiles and lariat ethers: dependence on pH and chain length

Lariat ethers and hydraphiles have been studied by the use of gel electrophoresis to evaluate the complexation of a 10 kilobase plasmid DNA. Both receptor systems show behaviour that reflects differences in side or spacer chain lengths. In addition, the formation of complexes or aggregates (identified by transmission electron microscopy, TEM) is pH dependent. Some detergents and polyamines can affect the formation and/or deaggregation of the complexes which TEM shows form particles of a relatively uniform size (~100–150 nm) and density for a specific receptor.

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

A prerequisite to the discovery and characterization of lipopolysaccharide (LPS) interaction with specific receptors and the resulting pathophysiological effects is the comprehensive structural analysis of LPS species. This brief review is aimed to summarize the use of gel electrophoresis linked to other biochemical technologies for detecting and characterizing LPSs. Lipopolysaccharide aggregates alone or mixtures containing LPSs and proteins/peptides can be separated by native agarose gel electrophoresis (NAGE), after which LPSs are detected with imidazole and zinc salts. A double-staining process with Coomassie brilliant blue R-250 enables the use of NAGE for detecting and studying protein-LPS interactions. For compositional analysis, the LPS aggregates are separated, with high resolution, by surfactant-polyacrylamide gel electrophoresis. After reverse staining with zinc-imidazole and elution from gel microparticles, glycoform-specific LPSs are ready for structural and biological analysis. For sequence analysis based on tandem electrospray ionization mass spectrometry (ESI-MS/MS) of oligosaccharides, LPSs are subjected to mild acid hydrolysis, dephosphorylation and permethylation. Also, O-deacylated LPS forms can be analyzed by matrix-assisted laser desorption/ionization-time of flight-MS. By comparison to spectra of unpurified LPSs, mass spectra of the micropurified LPSs show reduce heterogeneity and increased signal-to-noise ratios. Furthermore, the micropurification of LPSs prior to MS allows a higher sensitivity of detection for less abundant LPS glycoforms. The micropurified LPS fractions can be used to form self-assembled nanoaggregates which may be detected by dynamic light scattering. The effect of the O-side chain length on the Z-potential of LPS aggregates may be estimated by measurements based on laser Doppler electrophoresis. The thus obtained glycoform-specific LPSs are not only intact chemically but also biologically active as tested by e.g. Limulus amoebocyte lysate test, TNF-α assay and agonistic effect on human Toll-like receptor 4.

The application of pulsed field gel electrophoresis for molecular typing of causative agents of especially dangerous infections

The macro-restriction analysis of the microorganism DNA with the use of gel electrophoresis in pulsed field (PFGE typing, pulse electrophoresis) is applied in molecular biology to study the clonal structure and typing of causative agents of infectious diseases. Determining the degree of the relationship and definition of epidemiological interrelations of studied isolates, as well as studying the evolutionary history of pathogens, is performed by comparing DNA restriction patterns. This review presents an analysis of the use of the pulse electrophoresis in molecular-epidemiological research and the study of phylogeny of especially dangerous infections, cholera, and plague. The possibility of genetic heterogeneity of the Vibrio cholerae and Yersinia pestis populations is demonstrated; territorial and epidemiological characteristics of the spread of different isolate pulso-types, problems and prospects of the PFGE typing method in the system of epidemiological surveillance of cholera and plague in Russia are discussed.

Complementary mass spectrometric techniques for the quantification of the protein corona: a case study on gold nanoparticles and human serum proteins.

Once nanoparticles enter a biological system, it is known that their surface is instantly covered by the biomolecules present with preference to proteins. This protein corona has been a subject of numerous studies in order to reveal its composition. Besides that, growing interest exists in its quantitative determination in order to gain a deeper insight into the nature of these nanoparticle-protein bioconjugates. Only a few analytical methods are available nowadays, so the aim of this study is to provide a reliable and alternative methodology for the quantification of the protein corona. The suggested approach is based on the assumption that the total protein content within the corona can be correlated to its sulfur concentration due to the presence of cysteine and methionine as sulfur-containing amino acids. Once the most abundant proteins had been identified with the use of gel electrophoresis with subsequent peptide analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), the isolated nanoparticle-protein conjugates were subjected to total analysis of sulfur and the corresponding metal being present in the nanoparticles by inductively coupled plasma-mass spectrometry (ICP-MS). The concept is exemplarily demonstrated on citrate-stabilized gold nanoparticles (GNPs) incubated with human serum. Two different purification procedures were tested in order to isolate the sought bioconjugates. 26 most abundant proteins could be identified and an average of approximately 40 S atoms per protein was calculated and used for further studies. ICP-MS analyses of S/Au ratios served for the quantification of the protein corona revealing an absolute number of proteins bound to the incubated GNPs. Two main results could be obtained for this specific system under the chosen experimental conditions: the number of proteins per GNP decreased with their size from 10 nm to 60 nm and the obtained values suggested that the protein corona in this specific case was theoretically formed either as a monolayer (60 nm GNPs) or as a multilayer (5-7 protein layers per 10 nm GNP). Studies with bovine serum albumin (BSA) as the model protein showed similar results.

Use of gel electrophoresis and Raman spectroscopy to characterize the effect of the electronic structure of single-walled carbon nanotubes on cellular uptake.

It is well-known that the uptake of single-walled carbon nanotubes (SWNTs) by living cells depends on factors such as SWNT length and surface chemistry. Surprisingly, little is known about whether the electronic structure of a SWNT influences uptake. One reason for this has been the lack of methods to measure the uptake of SWNTs by cell populations. Previously, we developed a rapid, sensitive, and label-free sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) method for measuring the amount of SWNTs in lysates prepared from cultured cells (Wang et al. Anal. Chem.2009, 81, 294419296592). Herein, we describe the use of SDS-PAGE and microprobe Raman spectroscopy to detect and distinguish the electronic structure of SWNTs internalized by mammalian cells. Using normal rat kidney (NRK) cells and SWNTs dispersed with bovine serum albumin (BSA), we demonstrate that the method can detect both metallic and semiconducting SWNTs in lysates of cells that had internalized BSA-SWNTs and that the uptake of BSA-SWNTs by NRK cells is not influenced by SWNT electronic structure.

Inositol pyrophosphates: between signalling and metabolism

The present review will explore the insights gained into inositol pyrophosphates in the 20years since their discovery in 1993. These molecules are defined by the presence of the characteristic 'high energy' pyrophosphate moiety and can be found ubiquitously in eukaryotic cells. The enzymes that synthesize them are similarly well distributed and can be found encoded in any eukaryote genome. Rapid progress has been made in characterizing inositol pyrophosphate metabolism and they have been linked to a surprisingly diverse range of cellular functions. Two decades of work is now beginning to present a view of inositol pyrophosphates as fundamental, conserved and highly important agents in the regulation of cellular homoeostasis. In particular it is emerging that energy metabolism, and thus ATP production, is closely regulated by these molecules. Much of the early work on these molecules was performed in the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum, but the development of mouse knockouts for IP6K1 and IP6K2 [IP6K is IP6 (inositol hexakisphosphate) kinase] in the last 5years has provided very welcome tools to better understand the physiological roles of inositol pyrophosphates. Another recent innovation has been the use of gel electrophoresis to detect and purify inositol pyrophosphates. Despite the advances that have been made, many aspects of inositol pyrophosphate biology remain far from clear. By evaluating the literature, the present review hopes to promote further research in this absorbing area of biology.

A new method for stranded whole transcriptome RNA-seq

This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).

Rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

Yellow head virus (YHV) is a highly virulent pathogen that has caused severe mortality in cultivated shrimp (Penaeus monodon and Penaeus vannamei) in Thailand. There are several technologies that are applied to detect YHV for further control of the disease. RT-PCR is currently widely used in the laboratory, but it has some disadvantages related to cost, time-consuming and complexity. An alternative assay combines RT with loop-mediated isothermal amplification (LAMP) that not only provides high specificity, sensitivity and rapidity, but is also cheaper and more suitable for field applications in shrimp aquaculture than the RT-PCR. RT-LAMP is performed under isothermal conditions with a set of four to six primers designed to recognize six to eight distinct target sequences, and it has been combined with a chromatographic lateral-flow dipstick (LFD) to detect LAMP amplified product, which avoids the use of gel electrophoresis. In this study, RT-LAMP for the detection of YHV was developed by isothermal amplification at 65 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min (time required approximately 55 min, excluding RNA extraction and preparation time). The detection limit of RT-LAMP-LFD was 0.1 pg RNA extracted from shrimp infected with YHV equivalent to the nested RT-PCR, and no cross reaction was observed with other common shrimp viral pathogens. The LAMP method described in this study showed a rapid, high sensitivity and specificity and it is recommended as user-friendly for diagnosis of YHV in the field.

English

  Molecular markers are fast, efficient and reliable in detecting distinct differences between genotypes at DNA level. In particular, polymerase chain reaction (PCR)-based markers are widely preferred for genotype characterization in diverse crop species, including maize. It is impossible, however, to obtain the entire information required in plant breeding and conservation programmes solely from a single marker system without integration with other technique(s). The current study was, therefore, initiated to compare results from random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers in combination with high resolution melt analysis for effectiveness in detecting genetic variability or similarity among selected QPM inbred lines. Eight fresh leaf samples (two per line) were collected from each line for DNA extraction. The genomic DNA extracts were subjected to polymerase chain reaction (PCR) and high resolution melt analyses which involved three RAPD primers and three SSR primer pairs using a Real-Time PCR System. The curves of the HRM analysis showed fairly similar melt patterns among the lines for both types of marker systems. Results obtained from the gel-electrophoresis and genetic distance measures for RAPD markers were also in agreement with the HRM analysis. In general, both marker systems in combination with HRM analyses were able to detect genetic variations within and between the inbred lines, thus justifying the potential use of HRM analysis and indicating the possibility of excluding the use of gel-electrophoresis at the early stage of screening of large number of samples of newly developed inbred lines in order to save time and resources.   Key words: Genetic variability, polymerase chain reaction, quality protein maize, random amplified polymorphic DNA, simple sequence repeats.

Separation of olive proteins by capillary gel electrophoresis

Olive proteins are not well known and there are still a lot of unknown information requiring further studies focused on the determination and characterization of these proteins. Despite the widely use of gel electrophoresis, this is the first time that capillary gel electrophoresis (CGE) is applied to separate proteins extracted from olive fruits. Seven common peaks were identified in the twenty olive varieties studied in this work. According to their migration times, these seven peaks could correspond to molecules with molecular masses of 11.0±0.4, 13.9±0.5, 16.3±0.8, 22.1±0.6, 30±1, 48±1, and 53±2kDa. All of the determined molecular masses could be attributed to proteins and four of them have been previously observed by SDS-PAGE. The electrophoretic profiles were also evaluated for their capability to differentiate olive varieties according to their presumed geographical origin. Results demonstrated that this method could successfully classify the studied olive varieties by its combination with multivariate chemometrics tools.

A sensitive signal-on electrochemical assay for MTase activity using AuNPs amplification

A sensitive and simple signal-on electrochemical assay for detection of Dam methyltransferase (MTase) activity based on DNA-functionalized gold nanoparticles (AuNPs) amplification coupled with enzyme-linkage reactions is presented. This new assay takes advantage of the steric hindrance of AuNPs and the electrostatic repulsion between the negative-charge phosphate backbones of DNA modified on the AuNPs and redox probe [Fe(CN)6]3−/4−. In this method, the self-assembled ssDNA on the electrode is hybridized with its complement ssDNA modified on AuNPs to form dsDNA AuNPs bioconjugates containing specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I. Then, the AuNPs approach to the electrode and result in blockage of electronic transmission. It is eT OFF state. In the presence of Dam MTase and Dpn I, the specific sequence is methylated and cleavaged, which in turn release the DNA modified AuNPs from the electrode surface allowing free exchange of electrons. It generates a measurable electrochemical signal (eT ON). Differential pulse voltammetry (DPV) is employed to detect the recover current, which is related to the concentration of the Dam MTase. This method is simple, sensitive, nonradioactive and without use of gel-electrophoresis, PCR or chromatographic separation. Under optimized conditions, a linear response to concentration of Dam MTase range from 0.2U/mL to 10U/mL and a detection limit of 0.12U/mL are obtained. Furthermore, our new assay is a promising method to detect Dam MTase in the Luria–Bertani (LB) medium, as well as to screen inhibitors or drugs for Dam MTase.

The ProteoRed MIAPE web toolkit: A User-friendly Framework to Connect and Share Proteomics Standards

The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. First, it can verify that the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Second, the toolkit can convert several XML-based data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing, or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at http://www.proteored.org/MIAPE/.

Initial studies on quantitative DNA induced oxidation by gel electrophoresis (GE)-ICP-MS

One of the most important consequences of oxidative stress is the induction of oxidation in DNA molecules. Permanent modification of genetic material resulting from “oxidative damage” incidents represents the first step involved in mutagenesis, carcinogenesis, and aging. Therefore, there is an urgent need for monitoring DNA oxidative damage in a quantitative way. For this purpose, the present work evaluates the use of gel electrophoresis (GE) on-line coupled to inductively coupled plasma-mass spectrometry (ICP-MS) to address induced oxidative events in plasmid DNA pBlueScript SK (2961 bp). Oxidative stress is induced in the samples by addition of Fe2+ and H2O2 through the Fenton reaction. After optimization of the set-up for large DNA fragments, the GE separation of the different oxidation products (as induced by the Fenton reaction) were followed by 31P+ monitoring with ICP-MS and compared with conventional slab gels. The main advantage with the proposed set-up is that the quantification of each resulting oxidation product could be performed using just an inorganic phosphate as internal standard.