1. Procédure pour la quantification d&#39;ADN <ol><li> Tournez sur l&#39;instrument. </li><li> Ouvrez le logiciel BioSpec-nano à partir du PC de bureau. </li><li> Cliquez sur l&#39;acide nucléique simple-ADNdb onglet (figure 1). </li><li> Le logiciel va établir la communication avec l&#39;instrument et d&#39;exécuter une séquence d&#39;initialisation. Cela permet de vérifier que l&#39;instrument effectue conformément aux spécifications. </li><li> Sélectionnez 0,7 ou 0,2 mm sur le curseur trajet optique. Cela sélectionne le trajet optique et tous les calculs sont basés sur le trajet optique sélectionné. </li><li> Suivant dans BioSpec nano-cliquez logiciel de configuration-Autowiping configuration. Sélectionnez 1 ou 2. </li><li> Pipeter 2 uL (pour 0,7 mm) H 2 O ou mémoire tampon sur le piédestal. </li><li> Cliquez vierge ou F6 pour la mesure de vide. Chaque fois qu&#39;une nouvelle trajet optique est sélectionné, il est nécessaire d&#39;enregistrer un blanc. </li><li> Pipeter 2 uL d&#39;échantillon sur le piédestal. </li><li> Cliquez sur Démarrer dans le logiciel ou poussez le bouton de démarrage de l&#39;instrument. La fenêtre supérieure se déplace pour prendre contact avec la goutte d&#39;échantillon. Clignote au xénon peuvent être observés. </li><li> Répétez le même échantillon 3 fois pour déterminer la reproductibilité. </li><li> Toutes les données enregistrées sont enregistrées au format. Bourgeon fichier. </li><li> Pour enregistrer au format pdf. Ou. Csv cliquez sur Enregistrer pdf / csv ou F9. Les résultats peuvent également être sauvegardées dans un fichier. Csv en utilisant la sélection appropriée dans l&#39;onglet Enregistrer. </li></ol> 2. Protocole pour la quantification des protéines <ol><li> Sélectionnez l&#39;onglet quantification des protéines (figure 2). </li><li> Entrez le e280 coefficient d&#39;extinction, si elle est connue. </li><li> Si E280 n&#39;est pas connu de calculer à l&#39;aide e280Calc. </li><li> Sélectionnez 0,7 ou 0,2 mm sur le curseur trajet optique. </li><li> Cliquez sur Configuration-Autowiping configuration. Sélectionnez 2 ou supérieur. </li><li> Pipette 3 pi (0,2 mm) H 2 O ou mémoire tampon sur le piédestal. </li><li> Cliquez vierge ou F6 pour la mesure de vide. </li><li> Pipette 3 uL d&#39;échantillon de la protéine sur le piédestal. </li><li> Cliquez sur Démarrer dans le logiciel ou poussez le bouton de démarrage de l&#39;instrument. </li><li> Répétez le même échantillon 3 fois pour déterminer la reproductibilité. </li><li> Toutes les données enregistrées sont enregistrées au format. Bourgeon de fichier </li><li> Pour enregistrer au format pdf. Ou. Csv cliquez sur Enregistrer pdf / csv ou F9. </li></ol> 3. Les résultats représentatifs: 1. Résultats pour la quantification d&#39;ADN La figure 3 montre les données à partir de mesures sur un échantillon typique d&#39;ADN double brin. La concentration et OD 260/280 ratio calculé sont également représentées. La figure 4 contient les résultats des tests de reproductibilité pour la quantification d&#39;ADN. Les données des figures 3 et 4 résultats montre sont hautement reproductibles comme en témoigne le faible écart type relatif de 0,26%. Note 1: Imprécision dans la quantification d&#39;ADN peuvent découler de la présence des composants du tampon qui peut absorber dans la région UV. Il est également nécessaire d&#39;utiliser une solution d&#39;échantillon homogène. Comme la quantité d&#39;échantillon étant pipette est seulement 1-2 uL, pipetage minutieux des volumes précis sans l&#39;introduction de toute bulle est nécessaire. 2. Résultats pour quantification des protéines La figure 5 représente l&#39;analyse la concentration de protéines pour BSA. La concentration en protéines pg / ml est basé sur la valeur e280. La figure 6 montre l&#39;analyse de reproductibilité. Il est évident d&#39;après l&#39;écart type relatif de 0,51% que le BioSpec nano-mesures de la concentration précisément - non seulement pour l&#39;ADN, mais aussi pour les protéines. Note 2: Facteurs influant sur ​​la concentration de protéines Détermination de la concentration de protéines précis est réalisable quand il ya un minimum d&#39;effets de l&#39;instabilité provoquée par le pH, tampons, la température ou d&#39;additifs. La plupart des protéines purifiées passe par différentes étapes de traitement, d&#39;où il est possible que la protéine peut contenir des agents tensioactifs ou d&#39;autres additifs qui peuvent interférer avec la formation de gouttelettes. Pour ces échantillons, en utilisant la cellule de 5 mm trajet optique de quartz est fortement recommandée. <img src="/files/ftp_upload/2699/2699fig1.jpg" alt="Figure 1" /> Figure 1. Fenêtre de sélection Analyte pour la quantification d&#39;ADN double brin <img src="/files/ftp_upload/2699/2699fig2.jpg" alt="Figure 2" /> Figure 2. Fenêtre de sélection d&#39;analyte pour la quantification des protéines non étiquetés <img src="/files/ftp_upload/2699/2699fig3.jpg" alt="Figure 3" /> Représentant des données Figure 3. Pour la quantification d&#39;ADN <img src="/files/ftp_upload/2699/2699fig4.jpg" alt="Figure 4" /> La reproductibilité des données Figure 4. Pour la quantification d&#39;ADN <img src="/files/ftp_upload/2699/2699fig5.jpg" alt="Figure 5" /> Figure 5. Données représentatives pour la quantification des protéines <img src="/files/ftp_upload/2699/2699fig6.jpg"alt = &quot;Figure 6&quot; /&gt; Figure 6. Données de reproductibilité pour la quantification des protéines

<ol>
	<li>Aisubel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Short Protocols in Molecular Biology. , (1999).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+and+predict+molar+absorption+coefficient+of+a+protein.">How to measure and predict molar absorption coefficient of a protein.</a> Protein Science. 4, 2411-2423 .</li></ol>

Suja Sukumaran, Ph.D. est un employé de Shimadzu qui fait l&#39;instrument présenté dans cet article.

Les nano-BioSpec a été conçu pour résoudre les problèmes actuels inhérents aux méthodes de quantification de l&#39;ADN, telles que des résultats incohérents et le travail manuel fastidieux. Chaque mesure ne prend que 3 secondes, permettant à un biologiste expérimenté moléculaire pour analyser plusieurs échantillons d&#39;environ 3-6 en une minute. La fonction d&#39;essuie-glace unique, non seulement améliore le débit de la mesure, il élimine également le besoin pour le nettoyage manuel des piédestaux, résultant en report négligeables entre les mesures. Par ailleurs, des tests en utilisant seulement 2 lingettes pour les échantillons d&#39;ADN contenant 1000 ng / uL, a indiqué report moins de 2 ng / mL. La disponibilité d&#39;une cellule de 5 mm trajet optique fournit une option pour analyser diluer l&#39;acide nucléique et des protéines des échantillons et s&#39;étend à l&#39;utilisation de cet instrument pour d&#39;autres applications.

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<td>BioSpec-nano</td>
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<td>Calf Thymus DNA</td>
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concentration determination of nucleic acids and proteins using the micro-volume bio-spec nano spectrophotometer

Nucleic Acid procédures de quantification ont considérablement progressé au cours des trois dernières décennies. De plus en plus, des biologistes moléculaires nécessitent cohérente petit volume d&#39;analyse d&#39;échantillons d&#39;acides nucléiques pour leurs expériences. Les nano-BioSpec fournit une solution potentielle aux problèmes de la inexactes, les résultats non reproductibles, inhérente à la quantification méthodes actuelles de l&#39;ADN, via l&#39;optique spécialisée et un détecteur sensible PDA. Le BioSpec-nano a aussi des fonctionnalités automatisées telles que le montage, la mesure et de nettoyage sont effectuées par l&#39;instrument, éliminant ainsi le placement fastidieuse, répétitive et incohérente de l&#39;élément de fibre optique et le nettoyage manuel. Dans cette étude, les données sont présentées sur la quantification de l&#39;ADN et de protéines, ainsi que sur la reproductibilité et la précision de mesure. Contact avec l&#39;échantillon automatisée et balayage rapide permet de mesurer en trois secondes, résultant en un débit excellent. L&#39;analyse des données est effectué en utilisant les fonctionnalités intégrées dans le logiciel. La formule utilisée pour calculer la concentration en ADN est la suivante: Concentration de l&#39;échantillon = DF · (OD260-OD320) · NACF (1) Où DF = facteur de dilution de l&#39;échantillon et NACF = facteur de concentration d&#39;acide nucléiques. Le facteur de concentration d&#39;acide nucléique est fixé en conformité avec l&#39;analyte sélectionné 1. Résultats de concentration en protéines peut être exprimée en pg / mL ou en moles / L en entrant e280 et les valeurs de poids moléculaire respectivement. Lorsque les valeurs des résidus pour Tyr, Trp et la cystéine (SS obligataires) sont entrés dans l&#39;onglet e280Calc, les valeurs du coefficient d&#39;extinction sont calculés comme e280 = 5500 x (résidus Trp) + 1490 x (résidus Tyr) + 125 x (cystéine SS obligataire) . La valeur E280 est utilisé par le logiciel pour le calcul de la concentration. En plus de détermination de la concentration des acides nucléiques et les protéines, les nano-BioSpec peut être utilisé comme un ultra micro-volume spectrophotomètre pour beaucoup d&#39;autres analytes ou comme un spectrophotomètre standard utilisant des cellules 5 mm trajet optique.

Watch this Scientific Journal Video about Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer at JoVE.com

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Cette communication présente des données sur la précision et la reproductibilité des BioSpec nano-UV-VIS spectrophotomètre pour ADNdb et la quantification des protéines. Même avec ultra-petits volumes (1 à 2 L), la reproductibilité est excellente, tandis que la fonction automatisée essuyant améliore le débit et les résultats de report minimale pour obtenir des résultats plus précis.

Détermination de la concentration des acides nucléiques et des protéines en utilisant le micro-volume Bio-spec Spectrophotomètre Nano

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

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38.9K vues.  Scientific Instruments. Cette communication présente des données sur la précision et la reproductibilité des BioSpec nano-UV-VIS spectrophotomètre pour ADNdb et la quantification des protéines. Même avec ultra-petits volumes (1 à 2 L), la reproductibilité est excellente, tandis que la fonction automatisée essuyant améliore le débit et les résultats de report minimale pour obtenir des résultats plus précis.

Vidéo: Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Détermination microvolume concentration de protéines en utilisant le spectrophotomètre NanoDrop 2000c

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

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Biologie

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristallisant protéines membranaires pour déterminer la structure à l&#39;aide mésophases lipidique

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

L&#39;importance de la concentration de protéines correct pour Cinétique et Détermination d&#39;affinité en analyse structure-fonction

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification des ADNdb utilisant le Hitachi F-7000 Spectrophotomètre de fluorescence et de PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop microvolume quantification des acides nucléiques

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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.

D&#39;analyser et de structures d&#39;acides nucléiques avec 3DNA

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

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.

Étiquetage spécifique de la séquence des acides nucléiques et des protéines avec Methyltransferases et le cofacteur Analogues

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

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Incorporation spécifiques résidus de noncanonical acides aminés en protéines de modèle à l&#39;aide d&#39;un<em&gt; Escherichia coli</em&gt; Système de transcription-traduction libre Cell-

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Filtration Isolement des acides nucléiques: Une méthode d&#39;extraction de l&#39;ADN simple et rapide

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Détermination des acides sialiques dans les échantillons de foie et de lait de type sauvage et<em&gt; CMAH</em&gt; Souris knock-out.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...