The authors have nothing to disclose.

1.加工可能PANI合成<ol><li> 250mL丸底フラスコ中で完全にクロロホルム60ml中のアニリン（1 mlの11ミリモル）に溶解します。 5分間600rpmで撹拌し、氷で0〜5℃に冷却します。これは通常、15〜20分（ 図2A）を取ります。 </li><li> 600rpmで攪拌しながら、ドデシルベンゼンスルホン酸ナトリウム（NaDBS）（て7.44 gの21ミリモル）丸底フラスコ中のアニリンの溶液に加えます。 </li><li> 20ミリリットルの水に過硫酸アンモニウム（APS）（3.072グラム、13.5ミリモル）を溶解し、ドロップワイズ30分かけて反応の過熱を防ぐため、それのすべてを追加します。 </li><li> 24のための時間0-5℃で反応を行い、それがさらに24時間室温に到達させます。 </li><li>この反応混合物は、最初は15分後に乳白色を回して観察し、2時間後、最終的には24時間（ 図2B-F）の後に暗緑色に暗褐色。 </li><li>ブフナー漏斗とPANI-NaDBSソリューションをフィルタリングします。 80 mLのクロロホルムと120ミリリットルの水と混合eparation漏斗（ 図2G）。 </li><li>水性の上清中の未反応NaDBSとAPSを残して、室温で24時間のためのソリューションをインキュベートし、分離漏斗から暗緑色PANIを集めます。 </li></ol> 2. PANI-プローブ混合とUV照射<ol><li>クロロホルム - 水でPANI液10倍に希釈（1：3 V / V）および微量遠心管中で15分間穏やかに振動することにより、プローブDNAオリゴヌクレオチドの6.4μmolので希釈したPANIの200μLを混ぜます。 </li><li> 2分間架橋剤中のUVの1200μJ/ cm 2のPANI-DNA溶液を照射します。 UV露光が示された量に制限されていることが重要です。紫外線に長時間さらさは、PANIとDNAの共有結合架橋に起因する可能性が高いPANI中の蛍光変化を、損ないます。 </li><li> 6分間の17000×gで遠心分離によってペレット錯体、およびリン酸緩衝生理食塩水（PBS）で洗浄します。再びペレット化し、PBS中に再懸濁します。 </li></ol> 3. HPANI-プローブのybridization <ol><li> PANI-プローブ複合体の200μlに核酸を100μMの相補的なDNAオリゴヌクレオチドを8μlを添加またはターゲット。 </li><li> 40で15分間混合液をロッキングすることによりハイブリダイゼーションを行います °C。 </li><li> 6分間の17000×gで遠心分離することによってPANI複合体をペレット化。 PBSで洗浄し、水に再懸濁します。 </li></ol> 4.排出定常の蛍光測定<ol><li> 250 nmで励起することにより、96ウェルマイクロプレートに異なる処理からPANIを追加し、270から850 nmの範囲の発光蛍光を測定します。 PANIのための発光ピークは、500nm付近に観察されるべきです。 </li></ol>ハイブリダイズした二重の5蛍光顕微鏡測定<ol><li> 48時間、40℃でホウケイ酸ガラスのカバースリップとドライにコートPANIをドロップします。 </li><li>乾燥したPANI膜の上にプローブ（100μMの8μl）を追加し、UV光（1200μJ/ cm 2のそれを照射</su2分間のp&gt;）。 </li><li> 48時間40℃でPANI-プローブPBSでフィルムや乾燥を洗ってください。 </li><li>標的核酸を添加することにより15分間ハイブリダイゼーションを行います。これは生物学的サンプルまたは制御目標オリゴヌクレオチド（100μMの8μL）である可能性があります。 PBS洗浄に従ってください。 </li><li> 500 nmのロングパスフィルタを用いて、40X倍率で蛍光画像を取得します。 </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. M., Elkodary, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+polyaniline+nanostructures+in+micellar+solutions.">Synthesis of polyaniline nanostructures in micellar solutions.</a> J Appl Polym Sci. 124 (4), 3200-3207 (2012).</li><li>Tsotcheva, D., Tsanov, T., Terlemezyan, L., Vassilev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Investigations+of+Polyaniline+Prepared+in+the+Presence+of+Dodecylbenzenesulfonic+Acid.">Structural Investigations of Polyaniline Prepared in the Presence of Dodecylbenzenesulfonic Acid.</a> J Therm Anal Calorim. 63 (1), 133-141 (2001).</li><li>Jia, 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=Polyaniline-DBSA/organophilic+clay+nanocomposites:+synthesis+and+characterization.">Polyaniline-DBSA/organophilic clay nanocomposites: synthesis and characterization.</a> Synthetic Met. 128 (1), 115-120 (2002).</li><li>Kim, B. -. J., Oh, S. -. G., Han, M. -. G., Im, S. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Polyaniline+Nanoparticles+in+Micellar+Solutions+as+Polymerization+Medium.">Preparation of Polyaniline Nanoparticles in Micellar Solutions as Polymerization Medium.</a> Langmuir. 16 (14), 5841-5845 (2000).</li><li>Scales, C. 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).

核酸のPANIベースのセンサーは、DNAやRNAと相互作用するために、水中でのポリマーの可溶化を必要とします。水中のPANI分散物は、以前8報告されているように、ミセルを形成する界面活性剤を使用して達成されます。 4-スルホフタル酸ドデシルエステルなどの他の陰イオン界面活性剤ここで用いられるNaDBSに加えて、ノニルフェノールエトキシレート、またはセチルトリメチルアンモニ...

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.

図2Aは、APSの添加前に、 すなわち 、重合プロセスの開始時に反応セットアップを取り込みます。ミセル形成は、ミセル界面におけるプロセス-PANI合成が起こる反応の最初のステップである。 図2Bは、5分後に乳白色の解決策を示しています。 APSの30分後、反応はやや褐色に変わり追加されます。 図2Cは、オリゴマーの形成?...

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.

核酸のポリアニリンベースのセンサ

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 ビュー.  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.

ビデオ: 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).

脂肪酸の同定で<em&gt;セレウス菌</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 ...

生化学

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.

オリゴマー形成可能な高純度のアミロイドベータ42とアミロイドベータ40ペプチドを、利回りテーラードHPLC精製プロトコル

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.

電圧クランプ<em&gt;アフリカツメガエル</em&gt;蛍光性の非天然アミノ酸を用いた卵母細胞

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.

酸に不安定な保護基を保持するのに適したNi-シッフ塩基錯体の使用条件の加水分解

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.

タンパク質フィルム赤外線電気化学 [NiFe] ヒドロゲナーゼによって H2酸化反応に関する研究の実証

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.

ペプチド濃縮と質量分析によるタンパク質ユビキチン化部位の検出

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.

脂質混合アッセイ用リポソームへの組換えショウジョウバエアラストシンの洗剤支援再構成

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.

核酸プローブを用いたヌクレアーゼ活性の運動スクリーニング

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.

フミン酸鉱石、DOC、加湿物質及び腐植物含有市販品中のフミン酸及びフルボ酸の定量

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.

サッカロミセス・セレビシエ・スプライセソソーム・サブコンプレックスの過渡性インターアクター検出のためのグラフィックスの利用

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

核酸のポリアニリンベースのセンサ

要約

さらに動画を探す

この動画の章

脂肪酸の同定で