Vorremmo ringraziare l&#39;assistenza tecnica di Ines Racke.

Parte 1: Preparazione della soluzione colorante CBB <ol><li> 60-80 mg di CBB G-250 sono sciolti in 1 litro di acqua bidistillata mescolando per 2-4 ore. Infine, 3 ml di HCl concentrato viene aggiunto alla soluzione blu scuro con mescolando per un altro minuto e conservati al buio per un uso successivo. La soluzione può essere conservata per settimane fino a diversi mesi senza perdere la sua efficienza colorazione. </li><li> HCl concentrato deve essere maneggiato con la massima cura al solito utilizzati sotto una cappa aspirante. La soluzione finale sarà di circa pH 2, per cui i guanti devono essere utilizzati e qualsiasi contatto con la pelle deve essere evitato. </li></ol> Parte 2: SDS-PAGE <ol><li> Aliquote appropriato di campioni di proteine ​​sono miscelati con il caricamento del buffer ad una concentrazione finale di 1x tampone di caricamento. Noi usiamo il caricamento del buffer 2x 125mm con Tris/H3PO4 (pH 7,5 a 25 ° C), 2mM EDTA, 4% SDS, 200mm DTT, 0,02% bromofenolo blu e il 50% glicerolo. Buffer di carico di altri per SDS-PAGE può essere utilizzato come bene. </li><li> Campioni di proteine ​​vengono riscaldate per circa 5 minuti prima di caricarla. Nel frattempo, la camera di elettroforesi su gel è pronto per la corsa. Noi usiamo NuPAGE prefabbricati 4-12% © Bis-Tris gel (Invitrogen) in un SureLock XCell ® Mini-Cell (Invitrogen) con MES-tampone come il tampone di corsa, ma qualsiasi altro gel elettroforesi e il sistema può essere utilizzato come bene. </li><li> Campioni di proteine ​​sono stati caricati sul gel elettroforesi e correre per 50 minuti a 220V. </li></ol> Parte 3: colorazione del gel <ol><li> La cassetta gel viene smontato e il gel messo in una scatola per le fasi successive di lavaggio. </li><li> Circa 100 ml di acqua bidistillata viene aggiunta al gel e riscaldato nel forno a microonde per 30 secondi. Riscaldamento deve essere interrotto prima della bollitura si verifica. La scatola con il gel viene posto su un agitatore per 3-5 min. e questa fase di lavaggio viene ripetuto due volte con acqua fresca. </li><li> Basta soluzione colorante CBB è aggiunto per coprire il gel nella casella e la casella riscaldato nel forno a microonde per 10 secondi. senza portare all&#39;ebollizione. La scatola con il gel viene posto su un agitatore per la finitura della colorazione. Già dopo 1 minuto, bande proteiche possono essere osservati, dopo 15-30 min. la colorazione è abbastanza forte nella maggior parte dei casi. </li><li> La soluzione colorante è travasato e 50-100 ml di acqua bidistillata è aggiunto per Decolorare ulteriormente lo sfondo azzurro del gel in uno shaker. L&#39;acqua può essere sostituita con acqua fresca per ulteriori decolorazione, se necessario. </li><li> Il gel può essere scansionati, fotografati o essiccato per conservazione a lungo termine </li></ol> Parte 4: Risultati Rappresentante: Vedi fig. 1 per un gel colorato correttamente seguendo la procedura descritta. Vedi fig. 2 per un gel che non è stato lavato abbastanza a lungo prima di colorazione e colorazione residua SDS inibisce efficiente. Si noti che le corsie marcatore (*) contengono la stessa quantità di proteine ​​marker. <img src="/files/ftp_upload/1350/Fig1.png" alt="" /> Fig. 1: il gel colorato Rappresentante dopo aver caricato i campioni di una purificazione di proteine ​​(*: marcatore di peso molecolare). <img src="/files/ftp_upload/1350/Fig2.png" alt="" /> Fig. 2: CBB gel colorato che non è stato lavato abbastanza a lungo prima di colorazione CBB. Le bande di proteine ​​appaiono più deboli (si noti che la corsia * marcatore contiene la stessa quantità di proteine ​​come in fig. 1).

<ol>
	<li>Wondrak, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+for+fast+visualization+of+protein.">Process for fast visualization of protein.</a> US patent. , (2001).</li><li>Wondrak, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+for+fast+visualization+of+protein.">Solution for fast visualization of protein.</a> US patent. , (2001).</li></ol>

Nessun conflitto di interessi. La procedura descritta sopra è stato pubblicato originariamente nella domanda di brevetto da EM Wondrak (vedi rif.).

<ul><li> Le fasi di lavaggio sono fondamentali per la colorazione efficiente delle proteine. Una riduzione dei tempi di lavaggio di sotto di 2 minuti o ridotto volume di acqua (&lt;50 ml) può risultare in bande proteiche blu pallido, molto probabilmente a causa di una maggiore quantità di residui di SDS nel gel. </li><li> Se le proteine ​​stanno per essere analizzati mediante spettrometria di massa, le fasi di riscaldamento nel forno a microonde deve essere ignorata, il tempo per il lavaggio del gel estesa a circa 10 minuti in ogni passo ...

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<td>Coomassie Brilliant Blue G-250</td>
<td>&nbsp;</td>
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<td>any other CBB G-250 could be used as well</td>
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staining of proteins in gels with coomassie g-250 without organic solvent and acetic acid

Nei protocolli classica colorazione delle proteine ​​con Coomassie Brilliant Blue (CBB), soluzioni con alto contenuto di solventi organici tossici e infiammabili (metanolo, etanolo o 2-propanolo) e acido acetico vengono utilizzati per la fissazione, colorazione e decolorazione delle proteine ​​in un gel dopo SDS -PAGE. Per accelerare la procedura, il riscaldamento della soluzione colorante nel forno a microonde per un breve periodo è usato frequentemente. Questo di solito provoca l&#39;evaporazione di metanolo tossici o pericolosi, Etanolo o 2-propanolo e un forte odore di acido acetico in laboratorio, che dovrebbe essere evitato per motivi di sicurezza. In un protocollo originariamente pubblicato in due domande di brevetto da EM Wondrak (US2001046709 (A1), US6319720 (B1)), una composizione alternativa della soluzione di colorazione è descritta in cui nessun solvente organico o acido è usato. La CBB è disciolto in acqua bidistillata (60-80mg di CBB G-250 per litro) e 35 mM HCl è aggiunto come l&#39;unico altro composto nella soluzione colorante. Il staning CBB del gel è fatto dopo SDS-PAGE e completa il lavaggio del gel in acqua bidistillata. Riscaldando il gel durante il lavaggio e la colorazione fasi, il processo può essere completato più velocemente e senza compunds tossici o pericolosi stanno evaporando. La colorazione delle proteine ​​si verifica già entro 1 minuto dopo il riscaldamento il gel in soluzione colorante ed è completamente sviluppato dopo 15-30 minuti con uno sfondo blu che è leggermente decolorato completamente dal lavaggio prolungato del gel colorato in acqua bidistillata, senza alterare la proteina macchiata bande.

Watch this Scientific Journal Video about Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid at JoVE.com

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Un protocollo corto per la colorazione delle proteine ​​con Coomassie Brilliant Blue (CBB) G-250 in gel di poliacrilammide è descritto senza l&#39;utilizzo di solventi organici o acido acetico come nelle procedure di colorazione classica con CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

staining-proteins-gels-with-coomassie-g-250-without-organic-solvent

Research

JoVE Journal

Biology

52.8K Views.  EMBL Heidelberg. Un protocollo corto per la colorazione delle proteine ​​con Coomassie Brilliant Blue (CBB) G-250 in gel di poliacrilammide è descritto senza l'utilizzo di solventi organici o acido acetico come nelle procedure di colorazione classica con CBB.

Video: Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Colorazione proteine ​​in gel

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Ricerca

Biologia

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Veloce e sensibile colloidale Coomassie G-250 colorazione per le proteine ​​in gel di poliacrilammide

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profilatura di metiltransferasi e altri S-Adenosil- L-Omocisteina-binding proteine ​​mediante spettrometria di massa Compound Capture (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Esaminando la dinamica conformazionale delle proteine ​​di membrana In situ Con il sito-diretta Etichettatura fluorescenza

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

High-Throughput misurazione e classificazione di P organici in campioni ambientali

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantificazione delle proteine ​​Utilizzando Arricchimento Peptide immunoaffinità Accoppiato con spettrometria di massa

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 Their redox chemistry is even younger, just about ten years old, but despite this short history these deltahedral clusters ions E9n- (E = Si, Ge, Sn, Pb; n = 2, 3, 4) have already shown interesting and diverse reactivity and have been at the forefront of rapidly developing and exciting new chemistry.3-6 Notable milestones are the oxidative coupling of Ge94- clusters to oligomers and infinite chains,7-19 their metallation,14-16,20-25 capping by transition-metal organometallic fragments,26-34 insertion of a transition-metal atom at the center of the cluster which is sometimes combined with capping and oligomerization,35-47 addition of main-group organometallic fragments as exo-bonded substituents,48-50 and functionalization with various organic residues by reactions with organic halides and alkynes.51-58 
This latter development of attaching organic fragments directly to the clusters has opened up a new field, namely organo-Zintl chemistry, that is potentially fertile for further synthetic explorations, and it is the step-by-step procedure for the synthesis of germanium-divinyl clusters described herein. The initial steps outline the synthesis of an intermetallic precursor of K4Ge9 from which the Ge94- clusters are extracted later in solution. This involves fused-silica glass blowing, arc-welding of niobium containers, and handling of highly air-sensitive materials in a glove box. The air-sensitive K4Ge9 is then dissolved in ethylenediamine in the box and then alkenylated by a reaction with Me3SiC&equiv;CSiMe3. The reaction is followed by electrospray mass spectrometry while the resulting solution is used for obtaining single crystals containing the functionalized clusters [H2C=CH-Ge9-CH=CH2]2-. For this purpose the solution is centrifuged, filtered, and carefully layered with a toluene solution of 18-crown-6. Left undisturbed for a few days, the so-layered solutions produced orange crystalline blocks of [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en which were characterized by single-crystal X-ray diffraction. 
The process highlights standard reaction techniques, work-up, and analysis towards functionalized deltahedral Zintl clusters. It is hoped that it will help towards further development and understanding of these compounds in the community at large.

We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.

Sintesi di Nine-atomo ioni Deltahedral Zintl del germanio e la loro funzionalizzazione con gruppi organici

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

Analisi e costruzione di strutture acidi nucleici con 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Una procedura di screening chimico per glucocorticoidi segnalazione con un reporter del sistema Zebrafish Larva Luciferase

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

La colorazione delle proteine in gel con Coomassie G-250, senza solventi organici acido acetico e