Biz SAM formasyonu üzerinde yararlı tartışmalar için Melissa Grunlan kabul etmiş sayılırsınız. Bu araştırma, Robert A. Welch Vakfı (A-1585) tarafından kısmen desteklenmiştir.

Reaktiflerin hazırlanması BORU tamponu (pH 6,8 KOH ile ayarlanmış 80 mM BORU 1mm MgCl 2, 1mm EGTA) hazırlayın. -70 Ve mağaza kısım glukoz oksidaz, katalaz ve glikoz ° C ve taksol -20 ° C mevcut protokollere göre 1,2 Temsili miktar ve mağaza tubulin -70 ° C tedarikçi tarafından verilen talimatlara göre. Litografi kullanarak Desen Au Mikroelektronlar <ol><li> Bir katip veya gofret kesici kullanarak 1 cm 1.5 cm substratlar içine silikon gofret kesin. Kapak ve 5 dakika sonikasyon kadar aseton beher yerleştirerek substratlar temizleyin. Yüzeylerde kurumasına izin vermeden, taze aseton onları yıkayın, sonra hemen izopropanol ve kuru nitrojen içinde durulayın. </li><li> Au Mikroelektronlar temiz yüzeyler üzerinde istenen özellikleri boyutuna bağlı olarak, standart, pozitif tonu fotolitografi veya elektron demeti litografi kullanarak imal 3 desen geometri tasarlarken, kapsama sağlamak için her elektrot desen çapı 100 mikron altında tutmak PEG SAM (sonraki bölümde açıklandığı gibi). Yastık bağlayan bir elektrot (Şekil 1) ile 1 mm çizgi ile desen, 500 mm ile 300 konumlandırılmış temas pedleri (~ 300 mm 2) ekleyin . Akışını odasının montajı için desen etrafında yer kalmasına izin verecek yüzey ortasına yakın litografik kalıpları yerleştirin. <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="Şekil 1" /> Şekil 1. Örnek geometri. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1390/1390fig1.jpg">buraya tıklayınız</a> . </li><li> Karşı katmanı desen kalkınma sonra, karşı temas pedi cımbız kullanarak (Şekil 1) substrat kenarına bir çizgi çizebilir. Metal birikimi üzerine, sıfırdan elektrot ve yüzey kenarı arasındaki elektriksel temas sağlayacaktır. Biriktirme haznesi, tercihen iki kaynaktan ile bir metal katmanı yatırmak için kullanılır. Elektrotlar vakum bozmadan, Au 6 nm takip Cr 2 nm birikimi (yapışma) tarafından oluşturulur. Odasının içine donatılmış bir kuvars kristali mikro depolanması sırasında filmlerin kalınlığı ölçmek için kullanılabilir. </li></ol> SAM hazırlayın <ol><li> Onceden ve iyi havalandırılan bir alanda fırın - 75 ° C 250 ml cam beher 20 mL toluen ekleyerek silanization çözüm hazırlayın. Daha sonra plastik bir pipet kullanarak toluen için 1 ml PEG-silan (yani% 5 v / v) ekleyin. Yukarı ve aşağı pipetleme 5 ila 10 kat ve daha sonra 30 saniye boyunca pipet ile karıştırarak PEG-silan iyi karıştırın. </li><li> PEG-silan toluen çözümü tamamen sular altında ve eşit olarak beher alt kısmında aralıklı emin olun desenli örneklerin beher desenli yüzü yukarı bakacak şekilde yerleştirin. Fırında behere koyun ve 18 ila 21 saat fırında 75 ° C. SAM oluşturan iken, counterelectrode hazırlamak (aşağıya bakınız) ve kalan adımlar için gerekli olan tüm ek parçaları bir araya toplamak. </li><li> Fırında 18-21 saat sonra, toluen, PEG-silan viskoz bir kalıntı desenli örnekler bırakarak buharlaşır gerekir. 20 mL toluen beher ekleyin ve 1-2 dakika örnekleri bekletin. Örnekleri kaptan çıkarın ve sonra toluen, izopropanol durulayın ve nitrojen ile nihayet kuru. Bu noktada, numuneler temiz bir görünüm ve SAM katmanı çıplak gözle görünmez olmalıdır. SAMs serin kuru hava şartlarında (25 ° C, orta nem) altında saklanan iki gün kadar kullanılabilen bu şekilde hazırladı. Yüzeyinde bulutlu veya düzensiz Mutfak dekorasyonunun puf noktalari kalıntı kaplama Başarısız SAM oluşumuna neden olur. </li></ol> Counterelectrode Üretiyor Mikroelektrot depolanması için kullanılan aynı birikimi odasının kurulumu, 22 x 50 mm No 1 ya da 0 no&#39;lu cam lamel üzerine Au 4 nm Cr 1 nm, para yatırma bir counterelectrode olun. Counterelectrode açık gri ya da pembe bir renk tonu ile neredeyse şeffaf olmalıdır. Temiz tutmak için bir Petri kabındaki kaplı yan counterelectrode saklayın. MTS içine polimerize tubulin <ol><li> Çözülme tubulin ve rodamin tubulin etiketlenmemiş ve hemen elde etmek için parlak MTS etiketsiz için etiketli 1:2 oranında kombine. Birkaç kez aşağı yukarı pipetleme karıştırın. Isıtılmış su banyosunda 20-40 dakika tubulin polimerize 37 ° C. MTS uzunluğu uzun polimerizasyon kez daha büyük olacaktır. Tubulin polimerize olmasına rağmen, 20 mM için taksol BORU tampon içeren bir mikrosantrifüj tüpe ekleyerek BORU taksol tampon hazırlamak. Yukarı ve aşağı pipetleme birkaç kez ve vorteks taksol iyi karıştırın ve daha sonra 37 ısıtılmış banyoya tüp yeri ° C. Polimerizasyon sonra, sıcak BORU taksol tampon ve kalkan 1 / 100 MTS sulandırmakışıktan. Bu şekilde hazırlanan MTS zaman ve paket, daha fazla seyreltme azaltılabilir uzayacaktır. </li><li> 30 ml soğuk BORU tampon (0-4 ° C), 2-mercaptoethanol% 50, 5 ml katalaz glukoz oksidaz, 5 ml ve 5 ml glikoz 5 ml birleştirerek 10x oksijen süpürücü karışımı (OS) hazırlayın. 2 Glikoz son ilave edilmelidir. Her bileşen ekledikten sonra 3-5 kez pipetleme ve aşağı çevirerek karıştırın. OS karışımı buz koyun, yaklaşık 2 saat boyunca etkili olmaya devam edecektir. </li><li> MT polimerizasyon başarılı oldu ve MTS görüntüleme 1x OS karışımı ile hazırlanan MTS floresans uyarma birkaç dakika altında stabil olduğunu doğrulayın. Görüntüleme için bu şekilde hazırlanan MTS, rodamin floresan için uygun bir uyarma / emisyon filtre kombinasyonu kullandığınızdan emin olun. BORU 1x OS karışımı ile tampon ve görüntüleme kullanarak 1 / 10 ila 1 / 100 MTS seyreltilmesi görüntüleme bireysel filamentler için en iyi çalışma seyreltme belirleyin. </li><li> Bir akış odası, iki adet çift taraflı bant dışında 2-3 mm yerleştirilir büyük (22 x 50 mm) ve küçük (22 x 22 mm) cam lamel sandviç hazırlayın. Bant cam slayt üzerine yerleştirerek ve bir jilet kesim tarafından kolayca şeritler halinde kesilir. Mikropipet kullanarak akış hücre içine MTS bir kaç mikrolitre tanıtın. Yüksek büyütme görüntüleme için, uzun çalışma mesafesi hedefleri üst yüzey akış odasının görüntüleme için gerekli olabilir. Lamel / numune yüzey akış odasının üst yüzeyi arasındaki mesafe bant kalınlığına göre belirlenir ve 60-100 mm olmalıdır. </li></ol> Desen MTS elektroforez kullanarak <ol><li> Elektrot bant (Şekil 2) dik çalışan bağlantı yolu ile şeritler arasında olduğu gibi, desenli, SAM-kaplı numune üzerinde çift taraflı bant iki ince şeritler koyarak bir elektroforez odasının hazırlayın. Yaklaşık 3 mm (Şekil 2) tarafından örnek çıkıntılar counterelectrode kenarında çift taraflı bant, teyp gibi dokunmadan metal yüzü counterelectrode yerleştirin. Birkaç 15 cm uzunlukta, yalıtılmış bakır mıknatıs tel kesin ve her iki ucunda 1 cm yalıtım kapalı şerit. Counterelectrode (bu bir multimetre ile kontrol edilebilir) ile elektrik kontağı yapmaktan kaçınmaya dikkat çekerken, küçük bir damla gümüş boya kullanarak substrat maruz Au alan bir tel takın. Counterelectrode gümüş boya kullanarak ikinci bir tel takın. Teller gümüş boya ile bağlı bant ile örnekler daha güvenli olabilir. <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="Şekil 2" /> Şekil 2 ters bir floresan mikroskop Flow hücre montajı. Lütfen Şekil 2&#39;de büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1390/1390fig2.jpg">buraya tıklayın</a> . </li><li> 1x final konsantrasyon OS ile birlikte BORU tampon kullanarak istenilen seyreltme MTS tanıtın. 20X büyütme Görüntü ve bir voltaj uygulamadan önce silikon gofret desen bulmak. İsteğe bağlı olarak, akış odasının, akışkan akışı ve akış odasının düzlemde MT göçü önlemek için oje kullanılarak kapatılmalıdır. </li><li> 1 V kadar bir gerilim uygulayın ve MTS göç gözlemlemek. MTS, geri dönüşümlü hapsi istenen ise daha düşük voltaj (yaklaşık 0.5 V) kullanılıyor olabilir, ancak bu yanal kayma önlemek için akış odasının sızdırmazlık gerektirir. Tırnak cilası akış odasının açık biter sızdırmazlık için iyi çalışır. V 0.7 hakkında yukarıda gerilimler, bazı MT yapışma zayıf olsa, desen olabilir. Yaklaşık 0,9 V Yukarıdaki gerilimler, MT göçü daha hızlı olacak ve yapışma güçlü olacak. Mikroelektronlar yok etmek için de indükleyici elektroliz (gaz kabarcığı oluşumu) ve yaklaşık 1,2 V üstünde olasılığını önemli ölçüde artırır. Göç mikroskop odak dikey düzlemde ayarlayarak takip edilebilir. Doğru şekilde yapılmazsa, MTS elektrot yüzeylerinde lokalize. </li></ol>

<ol>
	<li>Rai-Choudhury, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).</li></ol>

The authors have nothing to disclose.

MTS, göç nedeniyle parlaklık ve düşük eşiğe kolayca floresan mikroskopi kullanılarak görüntülenmiştir. Sadece biyomoleküllerin tampon pH uygun ayarı ile net ücret taşımak için ikna olmasını gerektirir Bu yöntem, genellikle biyomolekülün desenlendirme için geçerli. Kapiler elektroforez kullanılan silika duvarlar gibi hiçbir ücret yüzeyleri vardır çünkü Electroosmosis Bu kurulum kaçınılmalıdır. Bu yöntem çeşitli değişiklikler yapılabilir. Sızdırma...

No: 1 borosilikat cam lamelleri (22 X 50 mm) VWR satın alınmıştır. Cilalı elektronik sınıf p-tipi &lt;111&gt; silikon gofret Yüksek saflıkta gümüş boya Yapısı Sonda, Inc (Katalog # 5001 AB) elde edilmiştir Addison Mühendisliği, Inc satın alındı. Yalıtımlı ince göstergesi Bakır Bobin Teli (~ 0.08 mm (0.003) çap), elektronik tedarik şirketlerinden elde edilebilir.

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

Biz, altın Mikroelektronlar, bir silikon taban poli (etilen glikol) (PEG) trimethoxysilane kendinden montajlı tek tabaka (SAM) oluşturmak için bir prosedür mevcut. PEG-SAM tek bir montaj adımda oluşturulur ve silikon ve altın yüzeyler üzerinde biyolojik kirlenmeye engeller. SAM standart, pozitif ton litografi desenli ceket Mikroelektronlar kullanılır. Mikrotübül bir örnek olarak kullanarak, geri dönüşümlü bir şekilde SAM-örtülü elektrot elektroforetik göç ikna etmek için bir DC gerilim uygulanır. Bir akış odası, görüntüleme için elektroforetik göç ve mikrotübül desenlendirme kullanılan<em&gt; In situ</em&gt; Epifloresans mikroskopi kullanılarak. Hedef moleküller hareketsiz ve böylece spesifik moleküler etkileşimler gerektirmez elektroforez çalışanı olarak bu yöntem, genellikle biyomolekülün desenlendirme uygulanabilir. Ayrıca, desen SAM moleküllerin doğrudan litografik teknikleri kullanarak çalışırken karşılaşılan sorunları önler. Elektron demeti litografi ile uyumluluk, bu yöntem nano ölçekli desen biyomoleküllerin kullanılmasına izin verir.

Watch this Scientific Journal Video about Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules at JoVE.com

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Biz altın Mikroelektronlar bir silikon taban üzerinde bir poli (etilen glikol), kendinden montajlı tek tabaka (PEG-SAM) oluşturmak için bir prosedür mevcut. PEG-SAM, tek bir adımda oluşan ve silikon ve altın yüzeyler biofouling engeller. Elektroforez sonra nano ölçekli desenlendirme biyomoleküllerin için kullanılır.

Desenlendirme Biyomoleküller Mikroelektronlar Kendinden monte mono tabakaları Zehirli

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

antifouling-self-assembled-monolayers-on-microelectrodes-for

Research

JoVE Journal

Biology

11.4K Görüntüleme.  Texas A&M University (TAMU). Biz altın Mikroelektronlar bir silikon taban üzerinde bir poli (etilen glikol), kendinden montajlı tek tabaka (PEG-SAM) oluşturmak için bir prosedür mevcut. PEG-SAM, tek bir adımda oluşan ve silikon ve altın yüzeyler biofouling engeller. Elektroforez sonra nano ölçekli desenlendirme biyomoleküllerin için kullanılır.

Video: Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Optik Şeffaf indiyum kalay oksit Elektrotlar Desenlendirme Hücreler

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Biyoloji

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Bio Flip-Chip kullanarak Embriyonik Kök Hücreler, Desenlendirme

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Yüzey immobilize Biyomoleküller Tek Molekül Floresans Ölçümler için Otomatik Sistem

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Yetişkin Fare kardiyomiyositlerde Tek Hücre transkripsiyonel Profilleme

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

Protein AKIL: A Tezgahı<em&gt; Siliko olarak</em&gt;<em&gt; De novo</emBiyomoleküllerin&gt; Tasarım

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

Elektroporasyon yoluyla Yaşam Mikroorganizmalar içinde içselleştirilmesi ve Floresan biyomoleküllerin Gözlem

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Belirli Genomik Bölgeler ve enChIP tarafından İlişkili Moleküllerin Tanımlanması İzolasyonu

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Siteye özel Protein lizin asetilasyon ve Succinylation veri bağımsız edinme kütle spektrometresi kullanılarak Stoichiometry miktar

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Senoterapötik Ilaçların tanımlanması için SA-β-Galactosidaz tabanlı tarama tahlili

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...