The authors have nothing to disclose.

1. İşlenebilir PANI Sentezi <ol><li> 250 mL&#39;lik yuvarlak dipli bir şişede kloroform, 60 ml tamamen (1 mi, 11 mmol) anilin içinde çözülür. 600, 5 dakika boyunca rpm&#39;de ve buz ile 0-5 ° C&#39;ye soğutun karıştırın. Bu, genellikle 15-20 dakika (Şekil 2A) alır. </li><li> 600 rpm&#39;de karıştırılırken, yuvarlak dipli bir reaksiyon kabına, anilin çözeltisine, sodyum dodesil benzen sülfonat (NaDBS) (7.44 gr, 21 mmol) ekleyin. </li><li> 20 ml su içindeki amonyum persülfat (APS) (3.072 gr, 13.5 mmol) çözündürülür ve tüm damla damla 30 dakika boyunca reaksiyon aşırı ısınmasını önlemek için ekleyin. </li><li> 24 saat süre ile 0-5 ° C&#39;de reaksiyonun yürütülmesi ve başka bir 24 saat süre ile oda sıcaklığına ulaşmasına izin verir. </li><li> Reaksiyon karışımı başlangıçta 15 dakika sonra süt beyazı edecek dikkat, 2 saat sonra, ve son olarak da 24 saat (Şekil 2B-F) sonra, koyu yeşil, sonra koyu kahverengi. </li><li> Buchner hunisi ile PANI-NaDBS çözelti filtre. olarak 80 ml kloroform ve 120 ml su ile karıştırılırAyırma hunisi (Şekil 2G). </li><li> Sulu süpernatan içinde reaksiyona girmemiş NaDBS ve APS bırakarak, oda sıcaklığında 24 saat boyunca çözelti inkübe ve ayırma hunisinden koyu yeşil Pani toplar. </li></ol> 2. PANI-prob Karıştırma ve UV Işınlama <ol><li> kloroform-su ile PANI çözeltisi 10x seyreltilir (1: 3 h / h) ile bir mikrofüj tüpü içinde, 15 dakika boyunca hafif sallanan probun DNA oligonükleotitleri 6.4 umol ile seyreltildi PANI 200 ul karıştırın. </li><li> 2 dakika boyunca, bir çapraz bağlayıcı UV 1200 μJ / cm2 olan PANI-DNA eriyiği ışın tedavisi. UV maruziyeti belirtilen tutarla sınırlı olduğu önemlidir. UV uzun süre maruz Bayan ve DNA kovalent çapraz bağlanma nedeniyle olası Pani floresans değişikliği ödün. </li><li> 6 dakika boyunca 17,000 x g&#39;de santrifüj ile pelet kompleksleri ve fosfat tamponlu tuzlu su (PBS) ile yıkanır. Yine pelet ve PBS içinde yeniden askıya. </li></ol> 3. HPani-prob ybridization <ol><li> Pani-prob komplekslerinin 200 ul nükleik asitleri 100 uM tamamlayıcı DNA oligonükleotidleri 8 ul ekle hedefler. </li><li> 40 ° C&#39;de 15 dakika boyunca çözelti karışımı sallanmasıyla hibridizasyon gerçekleştirmek ° C. </li><li> 6 dakika boyunca 17,000 x g&#39;de santrifüj ile PANI kompleksleri Pelet. PBS ile yıkayın ve su yeniden askıya. </li></ol> 4. Emisyon Kararlı Hal Floresan Ölçüm <ol><li> 250 nm&#39;de eksitasyon ile 96 oyuklu mikro-, farklı tedaviler Pani ekleyin ve 270-850 nm aralığında emisyon floresan ölçer. Pani bir emisyon tepe 500 nm civarında gözlenmelidir. </li></ol> Melezleştirilmiş Dubleks 5. Floresan Mikroskopi Ölçümü <ol><li> 48 saat süre ile, 40 ° C&#39;de bir borosilikat cam lamel üzerine kuru kaplama Pani bırakın. </li><li> Kurutulmuş PANI film üzerine prob (100 uM 8 ul) ilave edin ve UV ışığı (1.200 μJ / cm2 ile ışın </sup&gt;) 2 dakika karıştırıldı. </li><li> 48 saat süre ile, 40 ° C&#39;de PANI-prob PBS ile film ve kuru yıkayın. </li><li> Hedef nükleik asitleri ilave edilerek 15 dakika boyunca hibridizasyon gerçekleştirmek. Bu, bir biyolojik numune veya bir kontrol hedef oligonükleotit (100 uM 8 ul) olabilir. PBS yıkama izleyin. </li><li> 500 nm uzun geçiş filtresi ile, 40X büyütmede floresan görüntüleri elde edebilir. </li></ol>

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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).

Nükleik asitlerin bir PANI tabanlı sensör DNA ve RNA ile etkileşime girmesi için, su içinde polimerin çözünür hale gerektirir. Su içinde PANI dispersiyon, daha önce rapor edildiği gibi 8 miseller oluşturan, yüzey aktif maddeler kullanılarak gerçekleştirilir. 4-sülfoftalik asit dodesil ester gibi diğer anyonik yüzey aktif maddeler Burada kullanılan NaDBS ek olarak, nonil fenol etoksilat veya setiltrimetil amonyum bromür katyonik yüzey iyonik olmayan yüzey aktif maddeler de işlenebilir PANI 9,10 sentezi için kullanılabilir. 1 mol oranında: Burada tarif edilen sentez, 2 monomer aniline ilave anyonik sürfaktan NaDBS sabit bir oran ile başlar. yüzey aktif madde NaDBS yüksek hidrofil-lipofil dengesi (HLB) misel polimerik yağ damlacıkları ve su arasında denge kurularak kloroform-su içinde emülsifiye edilmiş anilin monomerinin polimerizasyonu sağlayan yer alır. gr miseller oluşturmak için 1.5 mol oranı: oksidanın APS sonra 1 ilave ediliriç kısım, bir çekirdeklendirici madde olarak hareket eder Pani, sayesinde. Bu, çevredeki çözücü 11,12 ile dinamik bir denge içinde Pani tutarken, monomerlerin kuyruk eki baş yol açar. Yüksek molekül ağırlıklı polimerler kadar kontrollü bir şekilde reaksiyon, 13 oluşturulur. Bu protokol, 50 nm ila 1,500 nm 3 e kadar olan parçacıkları oluşturur. PANI henüz yavaş büyür çözünür kalır bir tepki kuran kritik oksidan APS, düşük sıcaklık ve güçlü protonik asitler yokluğunda yavaş bir ektir. reaktan ait bazı optimizasyonu, iyi dağılmış Pani oluşturmak için gerekli olabilir. Bu yöntemle sentezlenebilir PANI bir emülsiyon olarak bir yıldan fazla stabildir. dispersiyon HLB dengesi bozulur edilebilir aşırı seyreltme PANI çökelmeye neden kaçınılmalıdır. Yüksek hızlı karıştırma çözeltisi içinde Pani tutmaya yardımcı olabilir. o pelet formati engel gibi mükemmel çözünürlük, arzu değildirbir emülsiyon esaslı sensör melezleme prosedürleri için önemli bir kriterdir üzerinde. Pani dağılmış oluşturduktan sonra, söz konusu DNA probu oligolar elektrostatik etkileşimi yoluyla polimer matrisine bağlanır. PANI imin grupları etkileşmesi DNA omurgası fosfatlar kendiliğinden meydana gelir. Non-spesifik PANI-nükleik asit etkileşimleri karmaşık biyolojik numunelerde 14 analit algılama zorlaştırıyor. Spontan etkileşimler ayırt edilebilir Bayan ve prob DNA arasındaki ilişki elde etmek için, kompleks bir UV ile ışınlandırılır. UV tetiklenen şarj lokalizasyonu ile oluşturulan polar kısımların oluşturulması polimer yüzeyinin 15 ıslatılabilirliğini arttırır. Optimum maruz yaklaşık 2 dakika, ve 500 nm&#39;de PANI floresans yaklaşık 5X (Şekil 3) yüksek olup olmadığını teyit edilebilir. Aşırı maruz kalma UV kompleksin PANI ve DNA, elektrostatik etkileşimler aksine, doe arasında çapraz bağlanma yol açabilirPANI floresans yükselmesine neden değil. sensörleri üreten süreci kolay ve hızlıdır. Polimerizasyon reaksiyonu, reaksiyona girmemiş oksitleyici ve NaDBS ayrı 24 saat, ardından 48 saat sonra tamamlanır. bir algılayıcı kullanılan miktardan çok büyük görece sentezi bu makalede protokolü tarafından üretilen hazır PANI miktar olarak seyrek gerçekleştirilir gerekir olan. Yüksek konsantrasyonlarda PANI emülsiyonu kararlı olduğu; Bununla birlikte, sonda takılması sırasında meydana gelen herhangi bir seyreltme sensör performansını tehlikeye, PANI çökelmesine neden olabilir. hedefleri tespit etmek PANI-prob komplekslerinin hemen kullanılması bu sorunu önlemek olacaktır. Birlikte, kompleksleri ve melezleşme yaratılması bir saatin altında yapılabilir. Pani-prob filmleri daha kararlı ve uzun süreli depolamadan sonra da kullanılabilir. Bu sensör teknolojisinin bir sınırlaması multiplexing&#39;i için son derece müsait olmamasıdır. Her tespit olayı ayrı bir tüp içinde gerçekleştirilmelidir. filmler olabilirfarklı prob bir filmin farklı bölgelerinde lekeli olabilir olarak çoklama için daha iyi bir biçim sağlar. Bir numune Noktası dizi üzerinde uygulanabilir ve her bir noktada floresans farklı gen ekspresyonunu değerlendirmek için kullanılır. Burada tarif edilen algılama mekanizması, diğer sistemlere göre pek çok avantaj sağlar. Pani kapsayan bir ayrışma mekanizması yoluyla nükleik asitleri tespit Raporlar DNA oligos 5 prob bağlı fluorophores güveniyor. Diğer stratejiler hedef nükleik asitler 16-17 tek baz-çift farkı ayırt başarısız problar, kovalent bağlanmasını kullanır. etiketleme veya bu teknolojinin algılama diğer ikincil modları ihmal elektro-aktif PANI diğer özelliklerini kullanmak için fırsatlar yaratır. Sensör tepkisi dolayısıyla sensörün eki-sıyrılma dinamikleri tarafından değiştirilmiş elektrokimyasal özelliklerinin ölçümü için uzun olabilir. Bu tekniği benimseyen Labs s bulacaksınızEnsor sistem, hızlı ve kolay yanı sıra maliyet etkili.

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.

Şekil 2A, APS ilave edilmeden önce polimerizasyon işlemi, örneğin, başlangıcında Reaksiyon setup yakalar. Misel oluşumu misel arayüzü süreç PANI sentezi meydana reaksiyonda ilk adımdır. Şekil 2B, 5 dakika sonra süt bir çözümü göstermektedir. APS Reaksiyon ilave edildikten sonra 30 dakika hafif kahverengi bir renge dönüşür. Şekil 2C oligomerler oluşumu ile bağlantılı bir renk değişikliği gösterir. Şekil 2B, uygun kısa zincirli PANI yüksek konsantrasyon gösteren, 4 saat sonra koyu kahverengi bir renk gösterir çok yavaş bir tepki. Son olarak kloroform-su (Şekil 2E) içinde dağılmış yüksek molekül ağırlıklı PANI koyu yeşil renkli bir çözelti elde edilebilir 48 saat sonra. Şekil 2F hiçbir faz ayrımı olmaksızın ayırma hunisinde iyi dağılmış, homojen PANI çözümü göstermektedir. (Şekil 2) ya da açılan kaplanmış filmlerin yüzeyi üzerinde (Şekil 3). Pani filmlerin taban floresans (Şekil 3A) göreceli olarak düşüktür. Prob, DNA film üzerine ilave edildi ve oda sıcaklığında kurumaya bırakılmıştır. Beklenen DNA PANI kompleks oluşturan floresan yoğun bir artış (Şekil 3b) gözlenebilir zaman. negatif yüklü DNA Elektron tutucu floresans artışa neden olan, PANI moleküllerinin yüksek elektron yoğunluğuna yol açar. Prob hedef çiftli hibridizasyonu neden ayrıldıktan sonra, PANI flüoresans taban yoğunluğuna (Şekil 3C) geri döner. Ayrılma melezleme üzerine DNA omurgasının uzlaşmanın PANI-prob etkileşim değişiklikleri nedeniyle oluşur. Bu teknoloji ile Algılama nükleik asitler, emülsiyon (Şekil 3D) &#39;de, doğrudan gerçekleştirilebilir. emülsiyon kullanmanın bir yararı<html> tabanlı sistem plaka okuyucu aletleri ile uyumluluk. Bu PANI floresan bir derece hassas ölçüm verir ve film kaplamada düzensizlikler nedeniyle zorluklar önler. emülsiyon biçiminde bir PANI tabanlı sensör son derece özeldir. Hedef oligo tek bir uyumsuzluğu (mm) giriş taban PANI floresan (Şekil 3D) geri başarısız olur. <img alt="Şekil 1" src="/files/ftp_upload/54590/54590fig1.jpg" /> UV ışınlarına maruz kalma aracılık prob Şekil 1. Ek PANI gelen dekolmanı hedef nükleik asitler sonuçlarının PANI floresan. Hibridizasyon artırır ve bazal floresan polimer geri dönmesinin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> iles / ftp_upload / 54590 / 54590fig2.jpg &quot;/&gt; Işlenebilen PANI Şekil 2. sentezi (A). Polimerizasyon reaksiyonunun başlangıcı. APS yavaş yavaş ilave edilir, 5 dakika sonra, (B). (C) 30 dakika sonra APS ekleme. 1 saat sonra (D). 4 saat sonra (D). (F) reaksiyon 48 saat sonra Yeşil ürün. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/54590/54590fig3.jpg" /> PANI Floresans Şekil 3. nükleik asit hibridizasyon tespit etmek için. 40 ° C&#39;de 48 saat boyunca kurutma, ardından borosilikat camına açılır kaplama ile oluşturulan (A) PANI film. Sonda immobilizasyon sonrası (B) PANI floresan. (C)PANI immobilize problar ile, hedef DNA&#39;nın hibridizasyonu takiben. Önce ve ya tamamlayıcı veya tek uyuşmazlığı (mm) hedef oligonükleotidler ile hibridizasyon sonrası PANI emülsiyon, 500 nm (D) Floresan. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54590/54590fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

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.

Nükleik Asitlerin bir Polyaniline tabanlı sensör

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.9K Görüntüleme.  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.

Video: 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).

Yağ Asitlerinin Tanımlanması içinde<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 ...

Biyokimya

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.

Bir Ismarlama HPLC Arıtma Protokolü That Oligomer Formasyonu Yetenekli Verim Yüksek saflıkta Amiloid Beta 42 ve Amiloid Beta 40 peptidler,

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.

Voltaj kelepçesi florometrisi<em&gt; Xenopus</emFloresan Doğal Olmayan Amino Asitler Kullanan Oositler

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.

Koruma Grupları aside dayanıksız Saklanmasına için uygun bir Ni-Schiff-Base hidrolizi Kompleksi kullanma Koşullar

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 kızılötesi elektrokimya H2 oksidasyon çalışma için [NiFe] Hydrogenase tarafından gösterdi

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.

Peptit Zenginleştirme ve Kütle Spektrometresi ile Protein Ubiquitinasyon Sitelerinin Saptanması

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.

Rekombinant Drosophila , lipid karıştırıcılar Için lipozomlar halinde deterjan destekli yeniden yapılanma

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.

Nükleik Asit Probları Kullanılarak Nükleaz Aktivitesinin Kinetik Taraması

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.

Humat Cevselerinde, DOC'ta, Humifik Malzemelerde ve Hümrik Madde İçeren Ticari Ürünlerde Humik ve Fulvik Asitlerin Nicelleştirilmesi

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.

Saccharomyces cerevisiae Spliceosome Subcomplexes Geçici Interactors Tespiti için Grafix kullanımı

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