Multiplex FISH and Spectral Karyotyping
Multicolor fluorescence in situ hybridization (mFISH) assays are indispensable for a precise description of complex chromosomal rearrangements and marker chromosomes. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole-chromosome painting (WCP) probes in multiplex-FISH (M-FISH) and spectral karyotyping (SKY). Here we present a review on the available mFISH approaches using WCP probes and describe a basic protocol for M-FISH and SKY. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Two- to Three-Color FISH
Two-color FISH is normally done to apply simultaneously a specific and a control probe. Three-color FISH can be done also as a combination of control and specific probes; however, also three specific target regions may be addressed at the same time. Here the application of commercially available two- and three-color FISH probes is described, and applications in diagnostics and research are discussed. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

FISH on Sperm, Spermatocytes and Oocytes
It is well known that chromosome in situ hybridization allows the unequivocal identification of targeted human somatic chromosomes. Different fluorescence in situ hybridization (FISH) techniques have been developed throughout the years, and following the mitotic studies, meiotic analyses have been performed using these different techniques. The application of FISH protocols on meiotic cells requires adaptation of standard protocols to the particularities of these cells. Specific sample fixation is usually required, and in some cases samples need to go through particular pretreatments to guarantee a successful FISH. The app...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Characterization of Archived Formalin-Fixed/Paraffin-Embedded or Cryofixed Tissue, Including Nucleus Extraction
Interphase cytogenetics using archival tissue samples is a straightforward approach to obtain“cytogenetic information” from nuclei of solid tissue samples. It is the major tool to investigate specific numerical chromosomal aberrations, chromosomal translocations, amplification of oncogenes, or deletion of tumor suppressor genes in archival tissue samples on a single-cell level, as no metaphase spreads can be obtained, obviously. Here a collection of protocols for archival sectioned and mounted material as well as extracted nuclei (from formalin-fixed/paraffin-embedded or cryofixed tissues) is presented. (Source...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Characterization of Mosaicism in Different Easy-to-Acquire Body Tissues Such As Buccal Smears, Skin Abrasions, Hair Root Cells, or Urine
Mosaic karyotypes are present in at least 0.3 to 1 % of clinical cases analyzed by banding cytogenetics. It is well known that the pattern of mosaicism can be extremely variant in different tissue types of the same patient. However, normally in maximum two different tissues of a child or an adult are cytogenetically studied, i.e., peripheral blood and skin fibroblasts. Here preparation protocols for four easily acquirable further tissues are presented. The resulting preparations of interphase nuclei can be used in routine interphase FISH applications. Here it is summarized how to treat buccal smears, skin abrasions,...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Tumorcytogenetic Diagnostics and Research on Blood and Bone Marrow Smears or Effusions
Blood smears, bone marrow smears, and effusions can be very helpful for quick molecular cytogenetic diagnostics in leukemia, lymphoma, or even solid tumors. Such samples can be studied by suited interphase FISH probes or probe sets for diagnostics, prognostics, therapeutic decisions, follow-up and control of effects of medication, or success of bone marrow transplantation. Here two protocols are provided on how to prepare smears or cell suspension in methanol/acetic acid (3:1) for subsequent FISH analyses. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

FISH in Uncultivated Amniocytes
In prenatal diagnostics, interphase FISH (iFISH) may be applied for the detection of certain numeric chromosomal aneuploidies. iFISH in uncultivated amniocytes can be performed from 12 weeks of gestation to the third trimester. Alternatively, the described aneuploidy screening set here is also suitable on short-term or uncultivated chorionic villus sampling (CVS) without metaphases as a fast screening test for the most common aneuploidies. Also this test helps to distinguish between culture-induced or real fetal triploidy or tetraploidy (mosaicism), which is found from time to time in the parallel cell culture; in this set...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Application of FISH to Previously GTG-Banded and/or Embedded Cytogenetic Slides
GTG-banded slides are often considered to be worthless for further molecular cytogenetic analyses. In this chapter we describe a simple protocol on how to reactivate such GTG-banded slides for further use in molecular cytogenetics. The slides can be previously Eukitt® or“Canada Balsam” embedded or previously not covered with a coverslip at all. With this possibility at hand, archival material of rare cytogenetic aberrations as well as more profound studies on actual cases with limited access to material can be performed. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Pre- and Postnatal Diagnostics and Research on Peripheral Blood, Bone Marrow, Chorion, Amniocytes, and Fibroblasts
Peripheral blood, amniocytic fluid, chorion, and fibroblasts are the most frequently used tissues for chromosome studies. All four of them are relatively easy to obtain and can simply be brought into short-term culture, and metaphase spreads can be prepared within rather short time. Such metaphase spreads, as well as the huge amounts of previously superfluous interphase nuclei in cytogenetic preparations, are a material very well suited for FISH analyses. All kinds of FISH probes can be used to analyze metaphase spreads, while the interphase nuclei actually can be analyzed for routine purposes only by satellite and locus-s...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

The Standard FISH Procedure
Molecular cytogenetics originally comprised of two basic approaches: fluorescence in situ hybridization (FISH) and primed in situ hybridization (PRINS). Nowadays FISH is the one routine approach still used in research and routine molecular cytogenetics field. Here the basic protocol in how to do FISH using commercial and/or homemade DNA probes is described. For a protocol for FISH on tissue sections, please refer to chapter by Thomas Liehr“ Characterization of Archived Formalin-Fixed/Paraffin-Embedded or Cryofixed Tissue, Including Nucleus Extraction .” Besides direct-labeled probes, also indirect-la...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

The Replicative Detargeting FISH (ReD-FISH) Technique in Studies of Telomere Replication
Based on the chromosome orientation-FISH (CO-FISH) procedure, the replicative detargeting FISH (ReD-FISH) was developed as a unique tool to study the replicative patterns of telomeres located on individual chromosomal arms. This method is also suited for examination of telomeres of species belonging to different classes of animals, for which well-proliferated cell cultures can be established and maintained. ReD-FISH is based on pulsed inclusion of labeled brominated nucleotides in replicating DNA, destruction of regions with brominated analogs, and standard FISH with single-strand-specific probe/probes. (Source: Springer p...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

RNA Imaging in Living Cells
Insight into the dynamics of RNA biosynthesis, processing, and cellular activity is highly valuable because it will deepen our understanding of cell physiology and help explain how mRNA-misregulation contributes to the development of many diseases. To date, the study of mRNA inside cells is a challenge; many analytical approaches focus only on quantifying expression levels of transcripts and are not capable of reporting their intracellular locations, which have emerged as a critical determinant of RNA function. Similarly, techniques capable of probing RNA localization merely offer snapshots in fixed cells. Herein, we descr...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Telomere Length Measurement by FISH
Telomere length influences numerous cellular processes such as senescence, carcinogenesis, and aging. Quantitative FISH (Q-FISH) is a comprehensive method that allows measuring of individual chromosome telomere length in single cell with the resolution of 200 base pairs. The method is based on the use of a peptide nucleic acid (PNA) telomere oligonucleotide probe and appropriate digital image software for capture and quantification of fluorescence signals. The length of telomere is directly related to its integrated fluorescence intensity, as PNA probes are assumed to hybridize quantitatively to telomeric repeats. For the ...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

One-Day Quick FISH
The classical FISH approach requires overnight incubation for proper hybridization result. Tissue morphological features are varying due to aggressive pretreatment and high temperatures applied. To increase the speed of the molecular cytogenetic finding and eliminate some of the technical limitations, an alternative one-day FISH method was recently introduced. This procedure allows the completion and evaluation of a FISH reaction within one day by the reduction of the hybridization time to 60–120 min. Moreover, the low denaturation temperature significantly contributes to better tissue and cell morphology of the...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Formamide-Free Fluorescence In Situ Hybridization (FISH)
Formamide is an ionising solvent which is widely used in molecular biology research for its thermodynamic effects on the DNA double-helix stability. In fluorescence in situ hybridization (FISH), the addition of formamide to aqueous buffers solutions of DNA enables key procedural steps—such as the prehybridization denaturation, the reannealing step and the post-hybridization stringency washes—to be carried out at lower, less harsh temperatures without compromising the overall efficiency and specificity of the hybridization. However, formamide is toxic and a potential teratogen, and its use in research laboratori...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

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...
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 spectrofluoro...
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

Metabolically Biotinylated Reporters for Electron Microscopic Imaging of Cytoplasmic Membrane Microdomains
We describe here the principles of this procedure and its application in the imaging of plasma membrane inner leaflet lipid rafts. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Luciferase Reporter Assays to Assess Liver X Receptor Transcriptional Activity
Luciferase reporter assays are sensitive and accurate tests that enable the analysis of regulatory sequences, the magnitude of transcriptional activity by transcription factors, and the discovery of gene regulatory elements and small-molecule modulators with high levels of precision. This is made possible through detection of bioluminescence produced by luciferase-coding reporters in a wide range of cellular environments. These assays are routinely used to analyze the activity of transcription factors, including the lipid-activated liver X receptor (LXR), in response to different stimuli as well as for the identification o...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Qualitative and Quantitative In Vitro Analysis of Phosphatidylinositol Phosphatase Substrate Specificity
Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Measurement of Long-Chain Fatty Acyl-CoA Synthetase Activity
Long-chain fatty acyl-CoA synthetases (ACS) are a family of essential enzymes of lipid metabolism, activating fatty acids by thioesterification with coenzyme A. Fatty acyl-CoA molecules are then readily utilized for the biosynthesis of storage and membrane lipids, or for the generation of energy by ß-oxidation. Acyl-CoAs also function as transcriptional activators, allosteric inhibitors, or precursors for inflammatory mediators. Recent work suggests that ACS enzymes may drive cellular fatty acid uptake by metabolic trapping, and may also regulate the channeling of fatty acids towards specific metabolic pathways. The ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Identification of the Interactome of a Palmitoylated Membrane Protein, Phosphatidylinositol 4-Kinase Type II Alpha
Phosphatidylinositol 4-kinases (PI4K) are enzymes responsible for the production of phosphatidylinositol 4-phosphates, important intermediates in several cell signaling pathways. PI4KIIα is the most abundant membrane-associated kinase in mammalian cells and is involved in a variety of essential cellular functions. However, the precise role(s) of PI4KIIα in the cell is not yet completely deciphered. Here we present an experimental protocol that uses a chemical cross-linker, DSP, combined with immunoprecipitation and immunoaffinity purification to identify novel PI4KIIα interactors. As predicted, PI4KII&alp...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Fluorescent Assays for Ceramide Synthase Activity
Ceramides are the central lipid metabolite of the sphingolipid family, and exert a potent influence over cell polarity, differentiation, and survival through their biophysical properties and their specific interactions with cell signaling proteins. Literature on the importance of ceramides in physiology and pathological conditions continues to grow, with ceramides having been identified as central effectors in major human pathologies such as diabetes and neurodegenerative conditions. In mammals, ceramide synthesis from a sphingoid base and a variable length fatty acid is catalyzed by a family of six ceramide synthases (CER...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Assaying Ceramide Synthase Activity In Vitro and in Living Cells Using Liquid Chromatography-Mass Spectrometry
Sphingolipids are one the major lipid families in eukaryotes, incorporating a diverse array of structural and signaling lipids such as sphingomyelin and gangliosides. The core lipid component for all complex sphingolipids is ceramide, a diacyl lipid consisting of a variable length fatty acid linked through an amide bond to a long chain base such as sphingosine or dihydrosphingosine. This reaction is catalyzed by a family of six ceramide synthases (CERS1-6), each of which preferentially catalyzes the synthesis of ceramides with different fatty acid chain lengths. Ceramides are themselves potent cellular and physiological si...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Assay for CDP-Diacylglycerol Generation by CDS in Membrane Fractions
CDP-DAG is a liponucleotide formed by the condensation of CTP with the phospholipid phosphatidic acid in a reaction catalyzed by CDP-DAG synthase (CDS). CDP-DAG is required for the synthesis of phosphatidylinositol; the parent molecule whence all seven phosphoinositides including the signaling molecules PI4P, PI(4,5)P2, and PI(3,4,5)P3 are derived. This protocol describes a highly sensitive radiometric assay to detect the generation of CDP-DAG on isolated biological membrane fractions. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Measuring Phosphatidylinositol Generation on Biological Membranes
Phosphatidylinositol (PI) is a phospholipid molecule required for the generation of seven different phosphoinositide lipids which have a diverse range of signaling and trafficking functions. The precise mechanism of phosphatidylinositol supply during receptor activated signaling and the cellular compartmentation of the synthetic process are still incompletely understood and remain controversial despite several decades of research in this area. The synthesis of phosphatidylinositol requires the activity of an enzyme called phosphatidylinositol synthase, also known as CDIPT, which catalyzes a reversible headgroup exchange re...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α 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 - November 12, 2015 Category: Cytology Source Type: news

Single-Molecule Imaging of Signal Transduction via GPI-Anchored Receptors
Lipid rafts have been drawing extensive attention as a signaling platform. To investigate molecular interactions in lipid rafts, we often need to observe molecules in the plasma membranes of living cells because chemical fixation and subsequent immunostaining with divalent or multivalent antibodies may change the location of the target molecules. In this chapter, we describe how to examine dynamics of raft-associated glycosylphosphatidylinositol (GPI)-anchored receptors and interactions of the receptors with downstream signaling molecules by single-particle tracking or single-molecule imaging techniques. (Source: Springer ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Analysis of the Phosphoinositide Composition of Subcellular Membrane Fractions
Phosphoinositides play critical roles in the transduction of extracellular signals through the plasma membrane and also in endomembrane events important for vesicle trafficking and organelle function (Di Paolo and De Camilli, Nature 443(7112):651–657, 2006). The response triggered by these lipids is heavily dependent on the microenvironment in which they are found. HPLC analysis of labeled phosphoinositides allows quantification of the levels of each phosphoinositide species relative to their precursor, phosphatidylinositol. When combined with subcellular fractionation techniques, this strategy allows measurement of ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Determination and Characterization of Tetraspanin-Associated Phosphoinositide-4 Kinases in Primary and Neoplastic Liver Cells
Accumulating evidence implicates phosphoinositide 4-phosphate as a regulatory molecule in its own right recruiting specific effector proteins to cellular membranes. Here, we describe biochemical and immunocytochemical methods to evaluate tetraspanin-associated phosphoinositide-4 kinases activity in primary human hepatic stellate cells (hHSC) and neoplastic hepatoblastoma cells. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Analysis of Sphingolipid Synthesis and Transport by Metabolic Labeling of Cultured Cells with [3H]Serine
Analysis of lipid biosynthesis by radioactive precursor incorporation provides information on metabolic rates and the identity of rate-limiting enzymes and transporters. The biosynthesis of sphingolipids in cultured cells is initiated in the endoplasmic reticulum (ER) by the formation of a sphingoid base from serine and palmitoyl-CoA. N-acylation of the sphingoid base produces ceramide, which is transported to the Golgi apparatus where phosphocholine or carbohydrate headgroups are added to form sphingomyelin (SM) and complex glycosphingolipids (GSLs), respectively. Herein is described a protocol to measure ceramide and SM ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Guidelines for the Use of Protein Domains in Acidic Phospholipid Imaging
Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs also known as phosphoinositides), phosphatidylserine (PS), and phosphatidic acid (PA). Acidic phospholipids are precursors of second messengers of key signaling cascades or are second messengers themselves. They regulate the localization and activation of many proteins, and are involved in virtually all membrane trafficking events. As such, it is crucial to understand the subcellular localization and dynamics of each of these lipids within the cell. Over the y...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

High-Throughput Fluorometric Assay for Membrane–Protein Interaction
Membrane–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 spectrofluorometer ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Analyzing Protein–Phosphoinositide Interactions with Liposome Flotation Assays
Liposome flotation assays are a convenient tool to study protein–phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein–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 liposome li...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Using Surface Plasmon Resonance to Quantitatively Assess Lipid–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 - November 12, 2015 Category: Cytology Source Type: news

Detection of Isolated Mitochondria-Associated ER Membranes Using the Sigma-1 Receptor
We describe in detail the preparation and purification of the MAM by using the sigma-1 receptor as the marker and demonstrate the uniqueness of this marker by using a variety of cells, peripheral and neuronal. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news

Isolation and Analysis of Detergent-Resistant Membrane Fractions
The hypothesis that the Golgi apparatus is capable of sorting proteins and sending them to the plasma membrane through “lipid rafts,” membrane lipid domains highly enriched in glycosphingolipids, sphingomyelin, ceramide, and cholesterol, was formulated by van Meer and Simons in 1988 and came to a turning point when it was suggested that lipid rafts could be isolated thanks to their resistance to solubilization by some detergents, namely Triton X-100. An incredible number of papers have described the composition and properties of detergent-resistant membrane fractions. However, the use of this method has also ra...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news