1. Verfahren zur DNA-Quantifizierung <ol><li> Schalten Sie das Gerät. </li><li> Öffnen Sie die BioSpec-nano-Software aus dem PC-Desktop. </li><li> Klicken Sie auf das Einfache Nucleic Acid-dsDNA Registerkarte (Abbildung 1). </li><li> Die Software wird die Kommunikation mit dem Gerät herzustellen und führen eine Initialisierung Sequenz. Dies bestätigt, dass das Gerät nach Angaben der Durchführung. </li><li> Wählen Sie 0,7 oder 0,2 mm über die Weglänge Schieberegler. Diese wählt die Weglänge und alle Berechnungen basieren auf der Weglänge ausgewählt wurden. </li><li> Next in BioSpec-nano-Software klicken Sie auf Setup-Autowiping Setup. Wählen Sie 1 oder 2. </li><li> Pipette 2 ul (für 0,7 mm) H 2 O oder Puffer auf dem Podest. </li><li> Klicken Sie auf leere oder F6 für Blank-Messung. Jedes Mal, wenn eine neue Schichtdicke gewählt wird, ist es notwendig, um einen leeren Datensatz. </li><li> Pipette 2 ul der Probe auf dem Podest. </li><li> Klicken Sie auf Start in der Software oder drücken Sie den Startknopf auf dem Instrument. Das obere Fenster bewegt, den Kontakt mit der Probe Tropfen zu machen. Xenon-Blitze beobachtet werden können. </li><li> Wiederholen Sie die gleiche Probe 3 mal um die Reproduzierbarkeit zu bestimmen. </li><li> Alle aufgezeichneten Daten ist. Bud-Datei gespeichert. </li><li> Zum Speichern als. Pdf oder. Csv-Datei klicken Sie auf Speichern pdf / csv oder F9. Die Ergebnisse können auch als. Csv-Datei, indem Sie die entsprechende Auswahl in der Registerkarte Speichern gespeichert werden. </li></ol> 2. Protokoll für Protein Quantifizierung <ol><li> Wählen Protein Quantifizierung Registerkarte (Abbildung 2). </li><li> Geben Sie den Extinktionskoeffizienten e280, falls bekannt. </li><li> Wenn e280 nicht bekannt ist, berechnen sie mit e280Calc. </li><li> Wählen Sie 0,7 oder 0,2 mm über die Weglänge Schieberegler. </li><li> Klicken Sie auf Setup-Autowiping Setup. Wählen Sie 2 oder höher. </li><li> Pipette 3 ul (für 0,2 mm) H 2 O oder Puffer auf dem Podest. </li><li> Klicken Sie auf leere oder F6 für Blank-Messung. </li><li> Pipette 3 ul der Proteinprobe auf dem Podest. </li><li> Klicken Sie auf Start in der Software oder drücken Sie den Startknopf auf dem Instrument. </li><li> Wiederholen Sie die gleiche Probe 3 mal um die Reproduzierbarkeit zu bestimmen. </li><li> Alle aufgezeichneten Daten ist. Bud-Datei gespeichert </li><li> Zum Speichern als. Pdf oder. Csv-Datei klicken Sie auf Speichern pdf / csv oder F9. </li></ol> 3. Repräsentative Ergebnisse: 1. Ergebnisse für die DNA-Quantifizierung Abbildung 3 zeigt die Daten aus Messungen an einem typischen doppelsträngige DNA-Probe. Die Konzentration und OD 260/280 Ratio berechnet werden ebenfalls gezeigt. Abbildung 4 enthält die Reproduzierbarkeit Testergebnisse für DNA-Quantifizierung. Die Daten in den Abbildungen 3 und 4 zeigt die Ergebnisse sind sehr gut reproduzierbar, wie aus den niedrigen relativen Standardabweichung von 0,26%. Hinweis 1: Ungenauigkeit in der DNA-Quantifizierung kann durch die Anwesenheit von Puffer-Komponenten, die im UV-Bereich absorbieren entstehen können. Es ist auch notwendig, um eine homogene Probe-Lösung verwenden. Da die Höhe der Probe pipettiert nur 1-2 ul, vorsichtiges Pipettieren von genauen Mengen ohne Einführung von Blasen ist nicht erforderlich. 2. Ergebnisse für Protein Quantifizierung Abbildung 5 zeigt Proteinkonzentration Analyse für BSA. Die ug / ml Proteinkonzentration ist auf der e280-Wert. Abbildung 6 zeigt die Reproduzierbarkeit Analyse. Daraus ist ersichtlich, dass die relative Standardabweichung von 0,51%, dass die BioSpec-nano-Konzentration misst präzise - nicht nur für DNA, sondern auch für Proteine. Hinweis 2: Faktoren, die die Protein-Konzentration Genaue Konzentration Bestimmung von Protein ist machbar, wenn es mindestens Effekte aus Instabilität, die durch pH, ​​Puffer, Temperatur oder Zusätze verursacht. Die meisten gereinigtes Protein durchläuft verschiedene Stufen der Verarbeitung, daher ist es möglich, dass das Protein Tenside oder andere Zusatzstoffe, die mit Tropfenbildung stören kann. Für solche Proben, mit dem 5 mm Schichtdicke Quarzküvette wird dringend empfohlen. <img src="/files/ftp_upload/2699/2699fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Analyte Auswahlfenster für doppelsträngige DNA-Quantifizierung <img src="/files/ftp_upload/2699/2699fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Analyte Auswahlfenster für nicht markiertes Protein-Quantifizierung <img src="/files/ftp_upload/2699/2699fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Repräsentative Daten zur DNA-Quantifizierung <img src="/files/ftp_upload/2699/2699fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Reproduzierbarkeit von Daten für die DNA-Quantifizierung <img src="/files/ftp_upload/2699/2699fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Repräsentative Daten für die Protein-Quantifizierung <img src="/files/ftp_upload/2699/2699fig6.jpg"alt = &quot;Bild 6&quot; /&gt; Abbildung 6. Reproduzierbarkeit von Daten für die Protein-Quantifizierung

<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 ist PhD Mitarbeiter von Shimadzu, der das Instrument in diesem Artikel vorgestellten macht.

Die BioSpec-nano wurde entwickelt, um aktuelle Themen innewohnenden DNA-Quantifizierung Methoden, wie inkonsistente Ergebnisse und mühsame Handarbeit zu lösen. Jede Messung dauert nur 3 Sekunden, so dass ein erfahrener Molekularbiologe ungefähr 3-6 mehrere Proben in einer Minute zu analysieren. Die einzigartige Scheibenwischer-Funktion verbessert nicht nur den Durchsatz der Messung, es entfällt auch die Notwendigkeit für die manuelle Reinigung der Container, die sich in vernachlässigbaren Verschleppung zwischen den Messungen. Darüber hinaus gaben Tests unter Verwendung von nur 2 Tücher für die DNA-Proben mit 1000 ng / ul, Verschleppung von weniger als 2 ng / ul. Die Verfügbarkeit einer 5 mm Schichtdicke Zelle enthält eine Option zum verdünnen Nukleinsäure-und Protein-Proben analysieren und erweitert den Einsatz dieses Instruments für andere Anwendungen.

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

Nucleic Acid Quantifizierung Verfahren haben sich in den letzten drei Jahrzehnten fortgeschritten. Mehr und mehr verlangen Molekularbiologen konsequent kleinvolumigen Analyse von Nukleinsäure-Proben für ihre Experimente. Die BioSpec-nano bietet eine mögliche Lösung für die Probleme der ungenauen, nicht reproduzierbare Ergebnisse, inhärent aktuellen DNA-Quantifizierung Methoden, über spezialisierte Optik und ein sensibles PDA-Detektor. Die BioSpec-nano hat auch automatisierte Funktionen, so dass Montage, Messung und Reinigung durch das Instrument fertig sind, wodurch langwierige, sich wiederholende und inkonsistent Platzierung der LWL-Element und manuelle Reinigung. In dieser Studie werden die Daten auf die Quantifizierung von DNA und Protein, sowie Mess-Genauigkeit und Reproduzierbarkeit vorgestellt. Automatisierte Probenvorbereitung Kontakt und schnelle Abtastung ermöglicht die Messung in 3 Sekunden, was zu einer hervorragenden Durchsatz. Die Datenanalyse erfolgt über die integrierten Funktionen der Software durchgeführt. Die Formel zur Berechnung DNA-Konzentration verwendet wird: Probenkonzentration = DF · (OD260-OD320) · NACF (1) Wo DF = Probe Verdünnungsfaktor und NACF = Nukleinsäure-Konzentration Faktor. Die Nukleinsäure-Konzentration Faktor ist in Übereinstimmung mit dem Analyten gewählt 1 gesetzt. Protein-Konzentration Ergebnisse können als pg / mL oder als mol / L durch Eingabe e280 und dem Molekulargewicht Werte ausgedrückt werden. Wenn Rückstände Werte für Tyr, Trp und Cystein (SS-Bindung) in der e280Calc Registerkarte eingegeben werden, sind die Extinktionskoeffizienten Werte wie e280 berechnet = 5500 x (Trp-Reste) + 1490 x (Tyr-Reste) + 125 x (Cystein SS-Bindung) . Die e280-Wert wird durch die Software für die Berechnung der Konzentration verwendet. Neben Konzentrationsbestimmung von Nukleinsäuren und Proteinen, kann die BioSpec-nano als ultra micro-Volumen Spektralphotometer für viele andere Analyten oder als Standard-Spektralphotometer mit 5 mm Schichtdicke Zellen verwendet werden.

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

Diese Mitteilung enthält Daten über die Genauigkeit und Reproduzierbarkeit der BioSpec-nano UV-VIS Spektralphotometer für dsDNA-und Protein-Quantifizierung. Selbst bei extrem kleinen Volumina (1 bis 2 L), ist die Reproduzierbarkeit ausgezeichnet, während die automatische Wisch-Funktion verbessert den Durchsatz und die Ergebnisse in minimaler Verschleppung für genauere Ergebnisse.

Konzentrationsbestimmung von Nukleinsäuren und Proteinen Mit dem Micro-Volumen Bio-spec Nano-Spektralphotometer

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

concentration-determination-nucleic-acids-proteins-using-micro-volume

Research

JoVE Journal

Biology

38.9K Aufrufe.  Scientific Instruments. Diese Mitteilung enthält Daten über die Genauigkeit und Reproduzierbarkeit der BioSpec-nano UV-VIS Spektralphotometer für dsDNA-und Protein-Quantifizierung. Selbst bei extrem kleinen Volumina (1 bis 2 L), ist die Reproduzierbarkeit ausgezeichnet, während die automatische Wisch-Funktion verbessert den Durchsatz und die Ergebnisse in minimaler Verschleppung für genauere Ergebnisse.

Serie: 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.

Mikrovolumen Proteinkonzentrationsbestimmung mit dem NanoDrop 2000c Spektrophotometer

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

Forschung

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.

Crystallizing Membranproteinen zur Strukturaufklärung mittels Lipidische Mesophasen

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.

Die Bedeutung der richtigen Protein-Konzentration für Kinetik und Affinity Bestimmung in Struktur-Funktions-Analyse

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.

Quantifizierung von dsDNA mit dem Hitachi F-7000 Fluoreszenz-Spektralphotometer und 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 Mikrovolumen Quantifizierung von Nukleinsäuren

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.

Analysieren und Bauen Nukleinsäurestrukturen mit 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.

Sequenzspezifische Markierung von Nukleinsäuren und Proteinen mit Methyltransferasen und Cofaktor-Analoga

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.

Rückstands spezifische Einbau nichtkanonischer Aminosäuren in Modellproteine ​​eine Verwendung<em&gt; Escherichia coli</em&gt; Zellfreie Transkription-Translation-System

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 Isolierung von Nukleinsäuren: Eine einfache und schnelle DNA-Extraktionsmethode

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.

Bestimmung von Sialinsäuren in Leber- und Milchproben von Wildtyp und<em&gt; CMAH</em&gt; Knock-out Mäuse.

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