1. Procedura per la quantificazione del DNA <ol><li> Accendere lo strumento. </li><li> Aprire il Biospec-nano software dal desktop del PC. </li><li> Clicca sul semplice Nucleic Acid-dsDNA scheda (Figura 1). </li><li> Il software stabilire una comunicazione con lo strumento ed eseguire una sequenza di inizializzazione. Questo verifica che lo strumento sta eseguendo in base alle specifiche. </li><li> Selezionare 0,7 o 0,2 mm sul cursore cammino ottico. Seleziona il cammino ottico e tutti i calcoli sono basati sul cammino ottico selezionato. </li><li> Prossima Biospec-nano click software di installazione-Autowiping installazione. Selezionare 1 o 2. </li><li> Pipettare 2 microlitri (per 0,7 mm) H 2 O o tampone sul piedistallo. </li><li> Clicca vuoto o F6 per la misurazione vuoto. Ogni volta che un nuovo cammino ottico è selezionata, è necessario registrare un vuoto. </li><li> Pipettare 2 ml di campione sul piedistallo. </li><li> Fare clic su Start nel software o premere il pulsante di avvio sullo strumento. La finestra superiore si muove per prendere contatto con la goccia del campione. Flash allo xeno possono essere osservati. </li><li> Ripetere lo stesso campione 3 volte a determinare la riproducibilità. </li><li> Tutti i dati registrati vengono salvati come file. Bud file. </li><li> Per salvare come file. Pdf o. Csv clicca salvare pdf / csv o F9. I risultati possono anche essere salvati come file. Csv utilizzando la selezione appropriata nella scheda Salva. </li></ol> 2. Protocollo per la quantificazione delle proteine <ol><li> Seleziona quantificazione scheda proteine ​​(Figura 2). </li><li> Inserire l&#39;E280 coefficiente di estinzione, se è nota. </li><li> E280 se non si sa calcolare usando e280Calc. </li><li> Selezionare 0,7 o 0,2 mm sul cursore cammino ottico. </li><li> Fare clic su Imposta-Autowiping installazione. Selezionare 2 o superiore. </li><li> Pipettare 3 ml (per 0,2 mm) H 2 O o tampone sul piedistallo. </li><li> Clicca vuoto o F6 per la misurazione vuoto. </li><li> Pipettare 3 ml di campione di proteine ​​sul piedistallo. </li><li> Fare clic su Start nel software o premere il pulsante di avvio sullo strumento. </li><li> Ripetere lo stesso campione 3 volte a determinare la riproducibilità. </li><li> Tutti i dati registrati vengono salvati come file. Bocciolo di file </li><li> Per salvare come file. Pdf o. Csv clicca salvare pdf / csv o F9. </li></ol> 3. Rappresentante dei risultati: 1. Risultati per la quantificazione del DNA La figura 3 mostra i dati da misure su un tipico esempio di DNA a doppio filamento. La concentrazione e OD 260/280 rapporto calcolato sono mostrati. Figura 4 contiene la riproducibilità dei risultati di prova per la quantificazione del DNA. I dati in figure 3 e 4 mostra i risultati sono altamente riproducibili, come risulta dalla bassa deviazione standard relativa del 0,26%. Nota 1: Inesattezza quantificazione del DNA possono derivare dalla presenza di componenti del tampone che possono assorbire nella regione UV. E &#39;anche necessario utilizzare una soluzione omogenea del campione. Poiché la quantità di campione in pipettato è solo 1-2 microlitri, pipettaggio attento di volumi precisi senza introduzione di eventuali bolle è richiesto. 2. Risultati per la quantificazione delle proteine La figura 5 rappresenta la concentrazione analisi delle proteine ​​per BSA. La mg / ml concentrazione di proteine ​​si basa sul valore E280. La figura 6 mostra l&#39;analisi riproducibilità. E &#39;evidente dalla deviazione standard relativa del 0,51% che la Biospec-nano misura la concentrazione di precisione - non solo per il DNA, ma anche per le proteine. Nota 2: Fattori che influenzano la concentrazione di proteine Determinazione accurata della concentrazione di proteine ​​è possibile quando ci sono effetti minimi di instabilità causata da pH, tamponi, la temperatura, o additivi. La maggior parte delle proteine ​​purificate passa attraverso varie fasi di lavorazione e quindi è possibile che la proteina può contenere tensioattivi o altri additivi che possono interferire con la formazione di goccioline. Per tali campioni, utilizzando il percorso ottico 5 millimetri cellula quarzo è altamente raccomandato. <img src="/files/ftp_upload/2699/2699fig1.jpg" alt="Figura 1" /> Figura 1. Analita finestra di selezione per la quantificazione del DNA doppio filamento <img src="/files/ftp_upload/2699/2699fig2.jpg" alt="Figura 2" /> Figura 2. Finestra di selezione per la mancata quantificazione Analita proteina marcata <img src="/files/ftp_upload/2699/2699fig3.jpg" alt="Figura 3" /> Dati Rappresentante Figura 3. Per la quantificazione del DNA <img src="/files/ftp_upload/2699/2699fig4.jpg" alt="Figura 4" /> Dati Riproducibilità Figura 4. Per la quantificazione del DNA <img src="/files/ftp_upload/2699/2699fig5.jpg" alt="Figura 5" /> Figura 5. Dati Rappresentante per la quantificazione delle proteine <img src="/files/ftp_upload/2699/2699fig6.jpg"alt = &quot;Figura 6&quot; /&gt; Figura 6. Riproducibilità dei dati per la quantificazione delle proteine

<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, PhD è un dipendente di Shimadzu che rende lo strumento descritto in questo articolo.

Il Biospec-nano è stato progettato per risolvere i problemi attuali inerenti i metodi di quantificazione del DNA, come i risultati inconsistenti e lavoro manuale noioso. Ogni misurazione dura solo 3 secondi, consentendo un esperto biologo molecolare per l&#39;analisi circa 3-6 campioni multipli in un minuto. La caratteristica unica del tergicristallo non solo migliora il throughput di misura, elimina anche la necessità di pulizia manuale dei piedistalli, con conseguente riporto trascurabile tra le misurazioni. Inoltre, i test utilizzando solo 2 salviette per i campioni di DNA contenente 1000 ng / mL, indicato riporto inferiore a 2 ng / mL. La disponibilità di una cella di 5 millimetri Lunghezza di percorso fornisce un&#39;opzione per analizzare diluire campioni di acidi nucleici e proteine ​​ed estende l&#39;uso di questo strumento per altre applicazioni.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BioSpec-nano</td>
<td>&nbsp;</td>
<td>Shimadzu</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calf Thymus DNA</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

concentration determination of nucleic acids and proteins using the micro-volume bio-spec nano spectrophotometer

Nucleic Acid procedure di quantificazione sono avanzati in modo significativo negli ultimi tre decenni. Sempre più, i biologi molecolari richiedono costante di piccoli volumi analisi dei campioni di acidi nucleici per i loro esperimenti. Il Biospec-nano fornisce una possibile soluzione ai problemi della inesatte, non riproducibili, inerenti gli attuali metodi di quantificazione del DNA, attraverso l&#39;ottica specializzati e un rivelatore sensibile PDA. Il Biospec-nano dispone anche di funzionalità automatizzate in modo tale che il montaggio, la misura e la pulizia sono fatte dallo strumento, eliminando così il posizionamento noioso, ripetitivo, e incoerente l&#39;elemento in fibra ottica e la pulizia manuale. In questo studio, i dati vengono presentati sulla quantificazione di DNA e proteine, nonché la riproducibilità di misura e precisione. Contatto automatizzata dei campioni e la scansione rapida consente di misurare in tre secondi, con conseguente rendimento eccellente. L&#39;analisi dei dati viene effettuata utilizzando il built-in caratteristiche del software. La formula utilizzata per calcolare la concentrazione del DNA è: Concentrazione campione = DF · (OD260-OD320) · NACF (1) Dove DF = fattore di diluizione del campione, NACF = fattore di concentrazione di acido nucleico. Il fattore di concentrazione di acido nucleico è impostato secondo l&#39;analita selezionato 1. Risultati concentrazione di proteine ​​può essere espressa come mg / ml o come moli / L inserendo E280 e valori di peso molecolare rispettivamente. Quando i valori di residui di Tyr, Trp e cisteina (SS obbligazioni) sono inseriti nella scheda e280Calc, i valori di estinzione coefficienti sono calcolati come E280 = 5500 x (residui Trp) + 1.490 x (residui Tyr) + 125 x (cisteina SS bond) . L&#39;E280 valore viene utilizzato dal software per il calcolo della concentrazione. Oltre alla determinazione della concentrazione di acidi nucleici e proteine, il Biospec-nano può essere usato come un ultra-micro volumi spettrofotometro per molti altri analiti o come uno spettrofotometro standard con 5 celle cammino ottico mm.

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

Questa comunicazione presenta i dati sulla precisione e la riproducibilità dei Biospec nano-UV-VIS per dsDNA e quantificazione delle proteine. Anche con ultra-piccoli volumi (da 1 a 2 L), la riproducibilità è eccellente, mentre la funzione automatica pulire migliora la velocità e si traduce in riporto minimo per ottenere risultati più precisi.

Determinazione concentrazione di acidi nucleici e proteine ​​Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano

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 Views.  Scientific Instruments. Questa comunicazione presenta i dati sulla precisione e la riproducibilità dei Biospec nano-UV-VIS per dsDNA e quantificazione delle proteine. Anche con ultra-piccoli volumi (da 1 a 2 L), la riproducibilità è eccellente, mentre la funzione automatica pulire migliora la velocità e si traduce in riporto minimo per ottenere risultati più precisi.

Video: 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.

Microvolumi Concentrazione Determinazione delle proteine ​​con il NanoDrop 2000c Spettrofotometro

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

Ricerca

Biologia

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.

Cristallizzare proteine ​​di membrana per la determinazione della struttura utilizzando Mesophases lipidica

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;importanza della concentrazione della proteina corretta per Cinetica e determinazione affinità nella struttura-funzione di analisi

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.

Quantificazione di dsDNA con il F-7000 Hitachi Fluorescenza spettrofotometro e 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 microvolumi quantificazione degli acidi nucleici

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.

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 ...

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 ...

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.

L&#39;incorporazione specifici residuo di non canonici amminoacidi in proteine ​​modello utilizzando un<em&gt; Escherichia coli</em&gt; Trascrizione-traduzione sistema non cellulare

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.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

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.

Determinazione degli acidi sialici nei campioni di fegato e latte di tipo selvatico e<em&gt; CMAH</em&gt; Mouse Knock-out.

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

Determinazione concentrazione di acidi nucleici e proteine ​​Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano

Determinazione concentrazione di acidi nucleici e proteine Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano