scholarly journals Label-free horizontal EMSA for analysis of protein-RNA interactions

2019 ◽  
Author(s):  
William Perea ◽  
Nancy L. Greenbaum
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dissociation Constant ◽  
Intermolecular Interactions ◽  
Ethidium Bromide ◽  
Fluorescent Labeling ◽  
Label Free ◽  
Cationic Protein ◽  
Intercalating Dye ◽  
Protein Components

AbstractWe describe a method to analyze the affinity and specificity of interactions between proteins and RNA using horizontal PAGE under non-denaturing conditions. The method permits tracking of migration of anionic and cationic biomolecules and complexes toward anode and cathode, respectively, therefore enabling quantification of bound and free biomolecules of different charges and affinity of their intermolecular interactions. The gel is stained with a fluorescent intercalating dye (SYBR®Gold or ethidium bromide) for visualization of nucleic acids followed by Coomassie® Brilliant Blue R-250 for visualizations of proteins; the dissociation constant is determined separately from the intensity of unshifted and shifted bands visualized by each dye. The method permits calculation of bound and unbound anionic nucleic acid and cationic protein components in the same gel, regardless of charge, under identical conditions, and avoids the need for radioisotope or fluorescent labeling of either component.

Mercury Electrodes in Nucleic Acid Electrochemistry: Sensitive Analytical Tools and Probes of DNA Structure. A Review

2004 ◽  
Vol 69 (4) ◽  
pp. 715-747 ◽  
Author(s):  
Miroslav Fojta
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Double Helix ◽  
Dna Structure ◽  
Electrochemical Analysis ◽  
Label Free ◽  
Helix Formation ◽  
Mercury Electrodes ◽  
Dna Strand ◽  
Dna Double Helix

This review is devoted to applications of mercury electrodes in the electrochemical analysis of nucleic acids and in studies of DNA structure and interactions. At the mercury electrodes, nucleic acids yield faradaic signals due to redox processes involving adenine, cytosine and guanine residues, and tensammetric signals due to adsorption/desorption of polynucleotide chains at the electrode surface. Some of these signals are highly sensitive to DNA structure, providing information about conformation changes of the DNA double helix, formation of DNA strand breaks as well as covalent or non-covalent DNA interactions with small molecules (including genotoxic agents, drugs, etc.). Measurements at mercury electrodes allow for determination of small quantities of unmodified or electrochemically labeled nucleic acids. DNA-modified mercury electrodes have been used as biodetectors for DNA damaging agents or as detection electrodes in DNA hybridization assays. Mercury film and solid amalgam electrodes possess similar features in the nucleic acid analysis to mercury drop electrodes. On the contrary, intrinsic (label-free) DNA electrochemical responses at other (non-mercury) solid electrodes cannot provide information about small changes of the DNA structure. A review with 188 references.

Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis

2005 ◽  
Vol 43 (8) ◽  
Author(s):  
Qing Huang ◽  
Wei-Ling Fu
Keyword(s):  
Comparative Analysis ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Ethidium Bromide ◽  
Gel Electrophoresis ◽  
Fluorescent Dyes ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Dna Staining

AbstractEthidium bromide (EB) is a mutagen and toxin that is widely used in the laboratory for visualization of nucleic acids. Safer nucleic acid stains, such as SYBR

Nanostructure-based surface-enhanced Raman scattering biosensors for nucleic acids and proteins

2016 ◽  
Vol 4 (10) ◽  
pp. 1757-1769 ◽  
Author(s):  
Jie Chao ◽  
Wenfang Cao ◽  
Shao Su ◽  
Lixing Weng ◽  
Shiping Song ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Raman Scattering ◽  
Nucleic Acids ◽  
Protein Detection ◽  
Surface Enhanced Raman Scattering ◽  
Label Free ◽  
Surface Enhanced ◽  
Surface Enhanced Raman ◽  
Enhanced Raman Scattering

Nanostructure-based SERS platforms have been developed for nucleic acid and protein detection ranging from label-free, labeled and multiplex analyses.

scholarly journals Analytical Ancestry: “Firsts” in Fluorescent Labeling of Nucleosides, Nucleotides, and Nucleic Acids

2009 ◽  
Vol 55 (4) ◽  
pp. 670-683 ◽  
Author(s):  
Larry J Kricka ◽  
Paolo Fortina
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Fluorescent Dyes ◽  
Fluorescent Labeling ◽  
Covalent Attachment ◽  
Detection Methods ◽  
Fluorescent Label ◽  
Nucleic Acid Detection ◽  
Fluorescent Labels ◽  
Fluorescent Properties

Abstract Background: The inherent fluorescent properties of nucleosides, nucleotides, and nucleic acids are limited, and thus the need has arisen for fluorescent labeling of these molecules for a variety of analytical applications. Content: This review traces the analytical ancestry of fluorescent labeling of nucleosides, nucleotides, and nucleic acids, with an emphasis on the first to publish or patent. The scope of labeling includes (a) direct labeling by covalent labeling of nucleic acids with a fluorescent label or noncovalent binding or intercalation of a fluorescent dye to nucleic acids and (b) indirect labeling via covalent attachment of a secondary label to a nucleic acid, and then binding this to a fluorescently labeled ligand binder. An alternative indirect strategy involves binding of a nucleic acid to a nucleic acid binder molecule (e.g., antibody, antibiotic, histone, antibody, nuclease) that is labeled with a fluorophore. Fluorescent labels for nucleic acids include organic fluorescent dyes, metal chelates, carbon nanotubes, quantum dots, gold particles, and fluorescent minerals. Summary: Fluorescently labeled nucleosides, nucleotides, and nucleic acids are important types of reagents for biological assay methods and underpin current methods of chromosome analysis, gel staining, DNA sequencing and quantitative PCR. Although these methods use predominantly organic fluorophores, new types of particulate fluorophores in the form of nanoparticles, nanorods, and nanotubes may provide the basis of a new generation of fluorescent labels and nucleic acid detection methods.

Nucleic Acid Molecules Prepared by Monolayer Techniques

1972 ◽  
Vol 30 ◽  
pp. 178-179
Author(s):  
Dimitrij Lang
Keyword(s):  
Electron Microscopy ◽  
Standard Deviation ◽  
Nucleic Acid ◽  
Cytochrome C ◽  
Nucleic Acids ◽  
Contour Length ◽  
Sample Standard Deviation ◽  
Molecular Weights ◽  
Length Measurements ◽  
Protein Monolayer

The success of the protein monolayer technique for electron microscopy of individual DNA molecules is based on the prevention of aggregation and orientation of the molecules during drying on specimen grids. DNA adsorbs first to a surface-denatured, insoluble cytochrome c monolayer which is then transferred to grids, without major distortion, by touching. Fig. 1 shows three basic procedures which, modified or not, permit the study of various important properties of nucleic acids, either in concert with other methods or exclusively:1) Molecular weights relative to DNA standards as well as number distributions of molecular weights can be obtained from contour length measurements with a sample standard deviation between 1 and 4%.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

Molecular insights on the dynamic stability of peptide nucleic acid functionalized carbon and boron nitride nanotubes

2021 ◽  
Vol 23 (1) ◽  
pp. 219-228
Author(s):  
Nabanita Saikia ◽  
Mohamed Taha ◽  
Ravindra Pandey
Keyword(s):  
Nucleic Acid ◽  
Boron Nitride ◽  
Nucleic Acids ◽  
Dynamic Stability ◽  
Peptide Nucleic Acid ◽  
Rational Design ◽  
Peptide Nucleic Acids ◽  
Boron Nitride Nanotubes ◽  
Molecular Hybrids ◽  
Single Wall Nanotubes

The rational design of self-assembled nanobio-molecular hybrids of peptide nucleic acids with single-wall nanotubes rely on understanding how biomolecules recognize and mediate intermolecular interactions with the nanomaterial's surface.

scholarly journals Characterization of Liposomes Using Quantitative Phase Microscopy (QPM)

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 590
Author(s):  
Jennifer Cauzzo ◽  
Nikhil Jayakumar ◽  
Balpreet Singh Ahluwalia ◽  
Azeem Ahmad ◽  
Nataša Škalko-Basnet
Keyword(s):  
Rapid Development ◽  
Fluorescent Labeling ◽  
Label Free ◽  
Batch Mode ◽  
Full Field ◽  
Quantitative Phase ◽  
Phase Microscopy ◽  
Quantitative Phase Microscopy ◽  
Tracking Ability

The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system’s instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QPM to localize subdiffraction-limited liposomes, estimate their size, and track their integrity over time. Moreover, QPM full-field-of-view images enable the estimation of a single-particle-based size distribution, providing an alternative to the batch mode approach. QPM thus overcomes some of the drawbacks of the conventional methods, serving as a relevant complimentary technique in the characterization of nanosystems.

Application strategies of peptide nucleic acids toward electrochemical nucleic acid sensors

The Analyst ◽  
2021 ◽  
Author(s):  
Qingteng Lai ◽  
Wei Chen ◽  
Yanke Zhang ◽  
Zheng-Chun Liu
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Peptide Nucleic Acids ◽  
Dna Probes ◽  
Highly Sensitive ◽  
Increased Sensitivity ◽  
Acid Sensors

Peptide nucleic acids (PNAs) have attracted tremendous interest in the fabrication of highly sensitive electrochemical nucleic acid biosensor due to their higher stability and increased sensitivity than common DNA probes....

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