The authors have nothing to disclose.

1. verarbeitbaren PANI Synthese <ol><li> Auflösen Anilin (1 ml, 11 mmol) vollständig in 60 ml Chloroform in einem 250 ml Rundkolben. Rühre bei 600 Umdrehungen pro Minute für 5 Minuten und Abkühlen auf 0-5 ° C mit Eis. Dies dauert in der Regel 15 bis 20 min (2A). </li><li> Hinzufügen Natriumdodecylbenzolsulfonat (NaDBS) (7,44 g, 21 mmol) zu der Anilin-Lösung in einem Rundkolben während bei 600 rpm gerührt wurde. </li><li> Man löst Ammoniumpersulfat (APS) (3,072 g, 13,5 mmol) in 20 ml Wasser und fügen Sie alle tropfenweise über 30 Minuten eine Überhitzung der Reaktion zu vermeiden. </li><li> Führen Sie die Reaktion bei 0-5 ° C für 24 Stunden, und lassen Sie es Raumtemperatur für weitere 24 Stunden zu erreichen. </li><li> Beachten Sie das Reaktionsgemisch zunächst drehen milchig weiß nach 15 min, dann dunkelbraun nach 2 h und schließlich zu dunkelgrün nach 24 h (2B-F). </li><li> Filtern Sie die PANI-NaDBS Lösung mit einem Büchner-Trichter. Mischen Sie mit 80 ml Chloroform und 120 ml Wasser in alseparation Trichter (2G). </li><li> Inkubiere die Lösung 24 h bei Raumtemperatur und sammle die dunkelgrüne PANI aus dem Scheidetrichter, so dass nicht-umgesetztes NaDBS und APS in dem wässrige Überstand. </li></ol> 2. PANI-Sonde Mischen und UV-Bestrahlung <ol><li> Verdünnen Sie PANI Lösung 10x mit Chloroform-Wasser (1: 3 v / v) und mischen Sie 200 ul verdünnt PANI mit 6,4 &amp; mgr; mol Sonden-DNA-Oligonukleotide durch sanftes für 15 min rocking in ein Mikrozentrifugenröhrchen. </li><li> Bestrahlen , um die PANI-DNA - Lösung mit 1200 &amp; mgr; J / cm 2 UV in einem Vernetzer für 2 min. Es ist kritisch, dass die UV-Belichtung mit der angegebenen Menge beschränkt ist. Verlängerte Exposition gegenüber UV kompromittiert die Fluoreszenzänderung in PANI, wahrscheinlich durch kovalente Vernetzung von PANI und DNA. </li><li> Pellet-Komplexe durch Zentrifugation bei 17.000 xg für 6 min und Waschen mit phosphatgepufferter Salzlösung (PBS). Pellet erneut und resuspendieren in PBS. </li></ol> 3. Hybridization von PANI-Sonde <ol><li> Aus 8 ul 100 uM komplementären DNA-Oligonucleotide oder Zielnukleinsäuren zu 200 ul PANI-Sonde-Komplexe. </li><li> Führen Hybridisierung durch Lösungsgemisch 15 min bei 40 rocking ° C. </li><li> Pellet die PANI-Komplexe durch Zentrifugation bei 17.000 xg für 6 min. Waschen mit PBS und Resuspendieren in Wasser. </li></ol> 4. Emissions Steady-State-Fluoreszenzmessung <ol><li> In PANI aus verschiedenen Behandlungen in einer 96-Well-Mikroplatte und messen Emissionsfluoreszenz im 270-850 nm-Bereich durch Anregung bei 250 nm. Ein Emissionsspitze für PANI sollte etwa 500 nm zu beobachten. </li></ol> 5. Fluoreszenzmikroskopie Messung der hybridisierten Duplex <ol><li> Drop-Mantel PANI auf einem Borosilikat-Deckglas und trocken bei 40 ° C für 48 Stunden. </li><li> In Sonde (8 ul 100 uM) auf einem getrockneten PANI Film und bestrahlen es mit UV - Licht (1200 &amp; mgr; J / cm 2 </sup&gt;) für 2 min. </li><li> Waschen Sie die PANI-Sonde Film mit PBS und trocken bei 40 ° C für 48 Stunden. </li><li> Führen Sie die Hybridisierung für 15 min von Ziel-Nukleinsäuren hinzufügen. Dies könnte eine biologische Probe oder eine Kontroll Zieloligonucleotid (8 ul 100 uM) sein. Folgen Sie mit einem PBS waschen. </li><li> Besorgen Sie sich die Fluoreszenzbilder bei 40-facher Vergrößerung, mit einem 500 nm Langpassfilter. </li></ol>

<ol>
	<li>Hahm, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+polymers+in+protein+detection+platforms:+optical,+electrochemical,+electrical,+mass-sensitive,+and+magnetic+biosensors.">Functional polymers in protein detection platforms: optical, electrochemical, electrical, mass-sensitive, and magnetic biosensors.</a> Sensors (Basel). 11 (3), 3327-3355 (2011).</li><li>Rahman, M. M., Li, X. B., Lopa, N. S., Ahn, S. J., Lee, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+DNA+hybridization+sensors+based+on+conducting+polymers.">Electrochemical DNA hybridization sensors based on conducting polymers.</a> Sensors (Basel). 15 (2), 3801-3829 (2015).</li><li>Sengupta, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+Intrinsic+Properties+of+Polyaniline+to+Detect+Nucleic+Acid+Hybridization+through+UV-Enhanced+Electrostatic+Interaction.">Utilizing Intrinsic Properties of Polyaniline to Detect Nucleic Acid Hybridization through UV-Enhanced Electrostatic Interaction.</a> Biomacromolecules. 16 (10), 3217-3225 (2015).</li><li>Song, E., Choi, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conducting+Polyaniline+Nanowire+and+Its+Applications+in+Chemiresistive+Sensing.">Conducting Polyaniline Nanowire and Its Applications in Chemiresistive Sensing.</a> Nanomaterials. 3 (3), 498 (2013).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyaniline+nanofibres+for+fluorescent+nucleic+acid+detection.">Polyaniline nanofibres for fluorescent nucleic acid detection.</a> Nanoscale. 3 (3), 967-969 (2011).</li><li>Oliveira Brett, A. M., Chiorcea, A. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy+of+DNA+Immobilized+onto+a+Highly+Oriented+Pyrolytic+Graphite+Electrode+Surface.">Atomic Force Microscopy of DNA Immobilized onto a Highly Oriented Pyrolytic Graphite Electrode Surface.</a> Langmuir. 19 (9), 3830-3839 (2003).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(m-Phenylenediamine)+Nanospheres+and+Nanorods:+Selective+Synthesis+and+Their+Application+for+Multiplex+Nucleic+Acid+Detection.">Poly(m-Phenylenediamine) Nanospheres and Nanorods: Selective Synthesis and Their Application for Multiplex Nucleic Acid Detection.</a> PLoS ONE. 6 (6), e20569 (2011).</li><li>Namgoong, H., Woo, D. J., Lee, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-chemical+structure+of+polyaniline+synthesized+by+self-stabilized+dispersion+polymerization.">Micro-chemical structure of polyaniline synthesized by self-stabilized dispersion polymerization.</a> Macromol Res. 15 (7), 633-639 (2007).</li><li>John, A., Palaniappan, S., Djurado, D., Pron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+preparation+of+solution+processable+conducting+polyaniline+by+inverted+emulsion+polymerization+using+didecyl+ester+of+4-sulfophthalic+acid+as+multifunctional+dopant.">One-step preparation of solution processable conducting polyaniline by inverted emulsion polymerization using didecyl ester of 4-sulfophthalic acid as multifunctional dopant.</a> J Polym Sci A: Polym Chem. 46 (3), 1051-1057 (2008).</li><li>El-Dib, F. I., Sayed, W. M., Ahmed, S. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corona-Stabilized+Interpolyelectrolyte+Complexes+of+siRNA+with+Nonimmunogenic,+Hydrophilic/Cationic+Block+Copolymers+Prepared+by+Aqueous+RAFT+Polymerization&#8224;.">Corona-Stabilized Interpolyelectrolyte Complexes of siRNA with Nonimmunogenic, Hydrophilic/Cationic Block Copolymers Prepared by Aqueous RAFT Polymerization&#8224;.</a> Macromolecules. 39 (20), 6871-6881 (2006).</li><li>Kadashchuk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+trions+in+conjugated+polymers.">Localized trions in conjugated polymers.</a> Phys Rev B. 76 (23), 235205 (2007).</li><li>Chang, H., Yuan, Y., Shi, N., Guan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+DNA+Biosensor+Based+on+Conducting+Polyaniline+nanotube+Array.">Electrochemical DNA Biosensor Based on Conducting Polyaniline nanotube Array.</a> Anal. Chem. 79, 5111-5115 (2007).</li><li>Zhu, N., Chang, Z., He, P., Fang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemically+fabricated+polyaniline+nanowire-modified+electrode+for+voltammetric+detection+of+DNA+hybridization.">Electrochemically fabricated polyaniline nanowire-modified electrode for voltammetric detection of DNA hybridization.</a> Eletrochim. Acta. 51, 3758-3762 (2006).</li></ol>

The work was supported by the University of Southern Mississippi College of Science and Technology and Mississippi College of Science and technology and Mississippi INBRE program (Award Number P204M103476 from the National Institute of general Medical Science).

A PANI-basierten Sensor von Nukleinsäuren erfordert die Solubilisierung des Polymers in Wasser, um mit DNA und RNA zu interagieren. Die Dispersion von PANI in Wasser Tenside erreicht Verwendung bilden Mizellen wie zuvor 8 berichtet. Neben den hier NaDBS andere anionische Tenside wie dodecylester von 4-Sulfophthalsäure, nicht - ionische Tenside wie Nonylphenolethoxylat oder kationische oberflächenaktive Mittel wie Cetyltrimethylammoniumbromid könnte auch für die Synthese von verarbeitbaren PANI 9,10<...

Conjugated polymers provide many options for molecular sensors. This includes fluorescence, electronic, and colorimetric responses1. There have been many efforts to incorporate conjugated polymers in nucleic acid sensors. However, most systems require secondary detection, limiting sensing options2. Recently, we reported a conjugated polymer-based sensor platform built on polyaniline (PANI) that exploits properties of this polymer, creating a label-free system3. PANI is an extensively conjugated electro-active polymer with properties such as fluorescence and resistance that are suitable for measuring biological systems4. The excitons within the structure are not localized leading to mobility of the positive charge between monomeric subunits. This provides a flexible scaffold of positive charges that can interact with the negatively charged backbone of DNA5,6. Importantly, electrostatically attached DNA is orientated such that nitrogenous bases can participate in base pairing. Association with DNA alters the electronic properties of PANI, an effect that can be enhanced by UV irradiation (Figure 1)3. Using this system, oligonucleotides complementary to target nucleic acids can be immobilized on PANI. Multiple studies have demonstrated that upon hybridization electrostatically adsorbed oligonucleotides dissociate from PANI or other cationic matrices due to conformational changes caused by the switch to a double-stranded DNA structure3,5,7.
In a sensor system where probe attachment modulates conjugated polymer properties, hybridization events can be transduced without labels or enzymatic modification of probes or target nucleic acids. Conjugated polymers offer great flexibility in detection methods, one of which is fluorescence. Through monitoring PANI fluorescence, concentrations of target nucleic acids as low as 10-11 M (10 pM) can be detected3. Detection is rapid, occurring within 15 minutes of hybridization, and specific where a single mismatch in a target molecule can be differentiated3. 
Fabrication of PANI-sensors is straightforward. High molecular weight PANI can be generated that is well-dispersed in water using standard synthesis procedures involving aniline monomer, surfactant, and controlled addition of an oxidant. Yield can be very high and unreacted oxidant removed by washing with water, ensuring no further PANI growth. PANI-probe association occurs spontaneously upon mixture, and complex formation is enhanced by mild UV exposure. Hybridization can be carried out immediately, and the changes in PANI fluorescence assayed following a short incubation. The simplicity of this technology makes it highly accessible to many laboratories.

<table border="0" cellpadding="0" cellspacing="0" width="1132">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">Aniline&#160;</td>
			<td style="width:131px;">Fisher Scientific&#160;</td>
			<td style="width:111px;">A7401-500&#160;</td>
			<td style="width:663px;">ACS, liquid, refrigerated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium peroxydisulfate</td>
			<td>Fisher Scientific&#160;</td>
			<td>A682-500&#160;</td>
			<td>ACS, crystalline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecylbenzene sulfonate</td>
			<td>Pfaltz &#38; Bauer&#160;</td>
			<td>D56340&#160;</td>
			<td>95% solid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>MCX 10601&#160;</td>
			<td>Liquid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA primers&#160;</td>
			<td>MWG operon&#160;</td>
			<td>n/a&#160;</td>
			<td>custom DNA sequence ~20bps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate&#160;</td>
			<td>USA Scientific&#160;</td>
			<td>1402-9800&#160;</td>
			<td>96 well, polypropylene as it is unreactive to chloroform</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate Adhesive Film</td>
			<td>USA Scientific&#160;</td>
			<td>2920-0000&#160;</td>
			<td>Reduces well-to-well contamination, sample spillage and evaporation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Cover Glass</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-544-D&#160;</td>
			<td>PANI coated on UV irradiated cover glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV crosslinker&#160;</td>
			<td>UVP&#160;</td>
			<td>HL-2000&#160;</td>
			<td>Energy: X100 &#956;J/cm2; Time: 2min</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hybridization Oven</td>
			<td>VWR&#160;</td>
			<td>01014705 T&#160;</td>
			<td>Temperature: 400C; with rocking for 15 min</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Apparatus&#160;</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Three necked round bottom flask for reaction; dropping funnel, stoppers, condenser, separating funnel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Leica Microsystems&#160;</td>
			<td>Leica IMC S80</td>
			<td>Magnification 20X; Pseudo color 536 nm; Exposure 86 ms; Gain 1.0X; Gamma 1.6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate Reader</td>
			<td>Molecular Devices&#160;</td>
			<td>89429-536</td>
			<td></td>
		</tr>
	</tbody>
</table>

a polyaniline-based sensor of nucleic acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

2A erfasst zu Beginn des Polymerisationsverfahrens des Reaktionsaufbau, also vor APS hinaus. Mizellenbildung ist der erste Schritt in dem Reaktionsverfahren-PANI - Synthese an der mizellaren Schnittstelle auftritt. 2B zeigt eine milchige Lösung nach 5 min. 30 min nach dem APS zugegeben wird die Reaktion dann auch in eine leicht braune Farbe. 2C zeigt die Farbänderung mit der Bildung von Oligomeren verbunden. 2D</strong...

Watch this Scientific Journal Video about A Polyaniline-based Sensor of Nucleic Acids at JoVE.com

A Polyaniline-based Sensor of Nucleic Acids

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

Ein Polyanilin basierenden Sensor von Nucleic Acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

a-polyaniline-based-sensor-of-nucleic-acids

Research

JoVE Journal

Biochemistry

8.0K Aufrufe.  University of Southern Mississippi. Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

Serie: A Polyaniline-based Sensor of Nucleic Acids

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

Identifizierung von Fettsäuren in<em&gt; Bacillus cereus</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

Forschung

Biochemie

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Ein Tailored HPLC Reinigungsprotokoll, dass die Renditen hochreine Amyloid Beta 42 und Amyloid Beta 40 Peptides, Fähig zur Oligomerbildung

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Spannklemme Fluorometrie in<em&gt; Xenopus</em&gt; Oozyten mit fluoreszierenden unnatürlichen Aminosäuren

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Hydrolyse eines Ni-Schiff-Base-Komplex unter Verwendung von Bedingungen, die für Retention von säurelabilen Schutzgruppen

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

Protein Film Infrarot-Elektrochemie für Studie der H2 Oxidation durch ein [NiFe] Hydrogenase unter Beweis gestellt

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Nachweis von Protein-Ubiquitinationsstellen durch Peptidanreicherung und Massenspektrometrie

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Waschmittelunterstützte Rekonstitution von rekombinantem Drosophila Atlastin in Liposomen für Lipid-Mischtests

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Kinetisches Screening der Nuklease-Aktivität mit Nukleinsäuresonden

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Quantification of Humic and Fulvic Acids in Humate Ores, DOC, Humified Materials and Humic Substance-Containing Commercial Products

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and shales, peats, composts and humic substance-containing commercial products. The method is based on the alkaline extraction of test materials, using 0.1 N NaOH as an extractant, and separation of the alkaline soluble humic substances (HS) from nonsoluble products by centrifugation. The pH of the centrifuged alkaline extract is then adjusted to pH 1 with conc. HCl, which results in precipitation of the HA. The precipitated HA are separated from the fulvic fraction (FF) (the fraction of HS that remains in solution,) by centrifugation. The HA is then oven or freeze dried and the ash content of the dried HA determined. The weight of the pure (i.e., ash-free) HA is then divided by the weight of the sample and the resulting fraction multiplied by 100 to determine the % HA in the sample. To determine the FA content, the FF is loaded onto a hydrophobic DAX-8 resin, which adsorbs the FA fraction also referred to as the hydrophobic fulvic acid (HFA). The remaining non-fulvic acid fraction, also called the hydrophilic fulvic fraction (HyFF) is then removed by washing the resin with deionized H2O until all nonabsorbed material is completely removed. The FA is then desorbed with 0.1 N NaOH. The resulting Na-fulvate is then protonated by passing it over a strong H+-exchange resin. The resulting FA is oven or freeze dried, the ash content determined and the concentration in the sample calculated as described above for HA.

This method provides a gravimetric quantification of humic substances (e.g., humic and fulvic acids) on an ash-free basis, in dry and liquid materials from soft coals (i.e., oxidized and non-oxidized lignite and sub-bituminous coal), humate ores and shales, peats, composts and commercial fertilizers and soil amendments.

Quantifizierung von Humin- und Fulvosäuren in Humatarzen, DOC, humifizierten Materialien und huminstoffhaltigen Handelsprodukten

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Verwendung von Grafix zum Nachweis transienter Interaktoren von Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Ein Polyanilin basierenden Sensor von Nucleic Acids

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