Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Cellular Delivery of Peptide Nucleic Acids (PNAs)
Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)–PNA conjugates and transfection of PNA–ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Rapid miRNA Imaging in Cells Using Fluorogenic Templated Staudinger Reaction Between PNA-Based Probes
Reactions templated by a specific nucleic acid sequence have emerged as an attractive strategy for nucleic acid sensing. The Staudinger reaction using an azide-quenched fluorophore and a phosphine is particularly well suited by virtue of its bioorthogonality and biocompatibility. The reaction is promoted by a complementary nucleic acid that aligns the phosphine with the azide-quenched fluorophore. Cellular RNAs can catalyze the Staudinger reaction and signal amplification can be achieved through multiple turnover of the template. Peptide nucleic acids (PNA) provide a convenient platform for the preparation of specific prob...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
PNA Fluorescent In Situ Hybridization (FISH) for Rapid Microbiology and Cytogenetic Analysis
Hybridization-based assays for the detection of nucleic acids including in situ hybridization are increasingly being utilized in a wide variety of disciplines such as cytogenetics, microbiology, and histology. Generally in situ hybridization assays utilize either cloned genomic probes for the detection of DNA sequences or oligonucleotide probes for the detection of DNA or RNA sequences. Alternately, PNA probes are increasingly being utilized in a variety of in situ hybridization assays [1, 2]. The neutral backbone of the PNA molecule allows for the PNA probes to bind to DNA or RNA under low ionic strength conditions that w...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Analysis of PNA Hybridization by Surface Plasmon Resonance
Reactions templated by a specific nucleic acid sequence have emerged as an attractive strategy for nucleic acid sensing. The Staudinger reaction using an azide-quenched fluorophore and a phosphine is particularly well suited by virtue of its bioorthogonality and biocompatibility. The reaction is promoted by a complementary nucleic acid that aligns the phosphine with the azide-quenched fluorophore. Cellular RNAs can catalyze the Staudinger reaction, and signal amplification can be achieved through multiple turnover of the template. Peptide nucleic acids (PNAs) provide a convenient platform for the preparation of specific pr...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Use of Peptide Nucleic Acids (PNAs) for Genotyping by Solution and Surface Methods
Peptide nucleic acids (PNAs) are synthetic oligonucleotide analogues based on a pseudopeptide backbone that bind complementary DNA or RNA with high affinity and specificity. In this chapter, three PNA-based genotyping assays are described: PCR clamping, fluorescence-based recognition, and microarray platform. The first two methods are performed in solution, while the microarray method uses a solid surface. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
DNA-Templated Native Chemical Ligation of Functionalized Peptide Nucleic Acids: A Versatile Tool for Single Base-Specific Detection of Nucleic Acids
Single base-specific detection of DNA/RNA sequences is of importance in the diagnosis of disease-associated genetic disorders or early stage cancer. This chapter introduces DNA-templated native chemical PNA ligation as a potentially useful tool for the sequence specific detection of nucleic acids. The template-induced alignment of PNA-thioesters and 1,2-aminothiol-PNAs in close proximity leads to an increase in their effective molarities. This facilitates PNA ligation to proceed at concentrations where no reaction would be possible in absence of the template. Moreover, hybridization of the rather short PNA conjugates with ...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
PNA Openers and Their Applications for Bacterial DNA Diagnostics
The unique ability of triplex-forming PNAs to invade the double helix has made it possible to develop a highly specific and sensitive approach for bacterial detection. The method uses short, about 20-bp-long, signature sequences presented as a single copy in the bacterial genome. Bacterial cells are fixed on slides and the PD-loop structure is assembled on the signature site with the help of PNA openers, which includes the circular probe. The sensitivity of the method is achieved via Rolling Circle Amplification (RCA) of the circular probe. The obtained amplicon is detected using short ssDNA decorator probes carrying fluor...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Zebrafish as a Model to Study Chemokine Function
Zebrafish have emerged as a powerful model organism to study embryo morphogenesis. Due to their optical clarity, they are uniquely suited for time-lapse imaging studies, providing insights into the dynamic processes underlying tissue formation and cell migration. These studies have been tremendously facilitated by the availability of transgenic zebrafish lines, labelling distinct embryonic structures, individual cells, or even subcellular structures, such as the nucleus. Zebrafish studies have revealed that the migration of several different cell types in the embryo is controlled by chemokines, small vertebrate-specific pr...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news
Unraveling Chemokine and Chemokine Receptor Expression Patterns Using Genetically Engineered Mice
Over the past 25 years, genetically engineered mouse models have become an integral and invaluable research tool to develop our understanding of mammalian physiology and pathology. This unit describes methods for generating transgenic mice, focusing on reporter animals relevant to chemokine receptor and ligand expression. Specifically, we describe the use of bacterial artificial chromosome (BAC) engineering and embryonic stem cell manipulation to generate “knock in” and transgenic mice. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news
A Novel Approach to Quantify G-Protein-Coupled Receptor Dimerization Equilibrium Using Bioluminescence Resonance Energy Transfer
Along with other resonance energy transfer techniques, bioluminescence resonance energy transfer (BRET) has emerged as an important method for demonstrating protein–protein interactions in cells. In the field of G-protein-coupled receptors, including chemokine receptors, BRET has been widely used to investigate homo- and heterodimerization, a feature of their interactions that is emerging as integral to function and regulation. While demonstrating the existence of dimers for a given receptor proved to be fairly straightforward, quantitative comparisons of different receptors or mutants are nontrivial because of inevi...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news
Chemokine Receptor Antagonist Development
This chapter describes assays that focus on the characterization of compounds identified in high-throughput screening campaigns, and the subsequent medicinal chemistry programs. They cover methods to determine potency in buffer, the effect of whole blood on the compounds’ activity and finally the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of the compounds in a rodent species. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news
Chemokine Receptor Interactions with Virus-Like Particles
Virus-like particles (VLPs) presenting conformational envelope proteins on their surface represent an invaluable tool to study molecular interactions between viruses and cellular receptors/co-receptors, eliminating biological risks associated with working with live native viruses. The availability of target cells expressing specific chemokine receptors facilitates the dissection of specific interactions between human immunodeficiency virus (HIV) viral envelope proteins and these receptors in the laboratory. Here, we describe a method to evaluate HIV-VLP binding to cellular chemokine co-receptors, by carboxyfluorescein succ...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news
Chemokine Receptors and Neural Stem Cells
Neural stem cells (NSCs) represent a limited population of progenitor cells in the central nervous system that sustain their self-renewal and multipotency from early development to adulthood. Recent evidence suggests that chemokine receptors are constitutively expressed by NSCs and are directly involved in stem cell biology. As cell surface receptors, chemokine receptors also provide an important avenue to enrich these cells and further identify the potential molecular pathways required to maintain their biological functions. Here, I describe in vitro methods that have been widely applied to sort, culture, maintain, and di...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news
Multispectral Imaging and Automated Laser Capture Microdissection of Human Cortical Neurons: A Quantitative Study of CXCR4 Expression
Quantifying protein and RNA expression within specific cell populations in vivo is an essential step in unraveling the complex mechanisms of neurological disease. The challenges associated with studying human brain tissue are commonly compounded by variations in postmortem interval, formalin fixation time, and tissue processing methods among others. The result is a sample population that is inherently heterogeneous, implying the need for reliable protocols that are sensitive to low levels of antigen while minimizing background and nonspecific staining. Here, we describe a single immunohistochemistry protocol on formalin-fi...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news
Chemokine-Dependent Signaling Pathways in the Peripheral Nervous System
Chemokines and their G-protein-coupled receptors play important roles in development, homeostasis, and the innate and adaptive immune response. Pathologic chemokine signaling pathways in the peripheral nervous system can be studied in peripheral nerves using human in vitro models of the blood–nerve barrier (BNB) and a reliable model of acute peripheral nerve inflammation called severe murine experimental autoimmune neuritis (EAN). This chapter describes a flow-dependent human leukocyte-BNB trafficking assay and the reliable induction of EAN in female SJL/J mice as tools to study pro-inflammatory chemokine-dependent s...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news
