Springer protocols feed by Cell Biology 
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.
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.FISH with and Without COT1 DNA
Complex FISH probes comprising large spans of genomic DNA always contain a high amount of dispersed repetitive sequences hampering the visualization of specific signals. To overcome this problem, different approaches have been elaborated that depend on experiment type and probe quality. A classical way to suppress repetitive sequences is to use unlabelled competitor DNA (sheared total genomic DNA or repeated sequences enriched DNA fractions). Here we present two protocols—the first one describes a rapid COT DNA isolation and peculiarities of its use in different FISH experiments, and the second is elaborated for COT-...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Microwave Treatment for Better FISH Results in a Shorter Time
Molecular cytogenetic approaches applying smaller, locus-specific probes like cDNA, plasmids, cosmids, fosmids, P1 clones, bacterial artificial chromosomes (BACs), or yeast artificial chromosomes (YACs) sometimes may be hampered by inefficient hybridization. Also, especially in diagnostics, FISH results may be required within a few hours. Here a FISH protocol using microwave treatment is presented, leading to better hybridization efficiency in case of smaller probes and evaluable results within a few hours. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Homemade Locus-Specific FISH Probes: Bacterial Artificial Chromosomes
Besides the well-known applications of bacterial artificial chromosomes (BACs) in classical molecular genetics, these probes are also well suited for molecular cytogenetic studies. BACs are nowadays the most often applied locus-specific probes in FISH. Various applications are possible like gene mapping, FISH banding, determination of chromosomal breakpoints, characterization of derivative chromosomes, studies on the interphase architecture, or the karyotypic evolution. Here the basic principle how BACs can be hybridized in situ on chromosome preparations is outlined. Moreover, an overview is given on possible questions to...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
FISH-Microdissection
FISH-microdissection (FISH-MD) is an approach combining FISH technique and chromosome microdissection in one experiment. This method enables reliable and straightforward identification of target chromosomes or chromosomal regions by FISH with specific probes and immediate microdissection of the chromosomal region of interest. FISH-MD can be applied when chromosome identification by trypsin-Giemsa staining/banding is complicated or not possible due to chromosomal morphology. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Generation of Paint Probes from Flow-Sorted and Microdissected Chromosomes
FISH with whole chromosome or region-specific painting probes made from either flow-sorted or microdissected chromosomes has revolutionized cytogenetics. Generation of paints from flow-sorted chromosomes relies on the use of an expensive and sophisticated fluorescence-activated cell sorter and suspensions of freshly prepared chromosomes. Preparation of paints from microdissected materials requires an inverted microscope with appropriate micromanipulators and metaphase chromosome spreads on coverslips. Painting probes made from flow-sorted chromosomes generally have better chromosomal coverage and can be used in a wide rang...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Commercial FISH Probes
Sources for commercially available FISH probes, labeled or unlabeled ones, are urgently necessary prerequisites of molecular cytogenetic field. Here some basics on commercially available probes are provided, and most commonly by such probes tracked loci in prenatal, postnatal, tumor, and pathology molecular cytogenetics are provided. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Classification of FISH Probes
Besides basic equipment, among consumables necessary for molecular cytogenetics, the choice of probes is the most critical point for a successful FISH experiment. Here the available FISH probes are reviewed and classified in different groups, i.e., according to their chemical properties, labeling, or target size. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Optical Filters and Light Sources for FISH
Brilliant fluorescence signals with almost no background and cross talk are the aim of FISH analysis in imaging systems. The precise selection of hardware components like optical filters and light sources plays a major role. Considering fluorescent dye characteristics is the base of configuring perfectly matched multicolor-FISH (mFISH) filters, which allow the simultaneous application of up to seven dyes. The spectral characteristics of filters are here explained with respect to microscope setups. Spectral cross talk, pixel-shift effects, and stable energy output will be the main issues in daily work. Specific hard-coated ...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Microscopy and Imaging
Microscopy is an integral part of fluorescence in situ hybridization (FISH) and related techniques. In combination with imaging technologies, microscopy has become a basis for a variety of FISH-based approaches to qualitative and quantitative molecular cytogenetic analysis of nucleic acids in situ. Here, these basic components of FISH—microscopy and imaging—are discussed. To avoid reproducing numerous textbooks dedicated to the fundamentals of microscopy (including the previous edition of this chapter in 2009) and manufacturer’s brochures about commercially available imaging systems, the basic aspects of ...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Background
The concept of molecular cytogenetics, its history, and perspectives are introduced here. FISH applications in clinical and tumor genetic diagnostics, including diagnostic guidelines and quality control, are reviewed. The impact of molecular cytogenetics in nowadays’ research is discussed, and finally a unique collection of internet pages is provided dealing with cytogenetics, molecular cytogenetics, as well as closely related fields. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news
Using Surface Plasmon Resonance to Quantitatively Assess Lipid & ndash;Protein Interactions
Surface Plasmon Resonance (SPR) is a quantitative, label-free method for determining molecular interactions in real time. The technology involves fixing a ligand onto a senor chip, measuring a baseline resonance angle, and flowing an analyte in bulk solution over the fixed ligand to measure the subsequent change in resonance angle. The mass of analyte bound to fixed ligand is directly proportional to the resonance angle change and the system is sensitive enough to detect as little as picomolar amounts of analyte in the bulk solution. SPR can be used to determine both the specificity of molecular interactions and the kineti...
Source: Springer protocols feed by Cell Biology - July 21, 2016 Category: Cytology Source Type: news
Analyzing Protein & ndash;Phosphoinositide Interactions with Liposome Flotation Assays
Liposome flotation assays are a convenient tool to study protein & ndash;phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein & ndash;lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposom...
Source: Springer protocols feed by Cell Biology - July 21, 2016 Category: Cytology Source Type: news
High-Throughput Fluorometric Assay for Membrane & ndash;Protein Interaction
Membrane & ndash;protein interaction plays key roles in a wide variety of biological processes. To facilitate rapid and sensitive measurement of membrane binding of soluble proteins, we developed a fluorescence-based quantitative assay that is universally applicable to all proteins. This fluorescence-quenching assay employs fluorescence protein (FP)-tagged proteins whose fluorescence intensity is greatly decreased when they bind vesicles containing synthetic lipid dark quenchers, such as N-dimethylaminoazobenzenesulfonylphosphatidylethanolamine (dabsyl-PE). This simple assay can be performed with either a spectrofluoromete...
Source: Springer protocols feed by Cell Biology - July 21, 2016 Category: Cytology Source Type: news
Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase & alpha; in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format
Lipid kinases are important regulators of a variety of cellular processes and their dysregulation causes diseases such as cancer and metabolic diseases. Distinct lipid kinases regulate the seven different phosphorylated forms of phosphatidylinositol (PtdIns). Some lipid kinases utilize long-chain lipid substrates that have limited solubility in aqueous solutions, which can lead to difficulties in developing a robust and miniaturizable biochemical assay. The ability to prepare the lipid substrate and develop assays to identify modulators of lipid kinases is important and is the focus of this methods chapter. Herein, we desc...
Source: Springer protocols feed by Cell Biology - July 21, 2016 Category: Cytology Source Type: news
Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes
We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
