우리는 SAM 형성에 도움이 토론에 대한 멜리사 Grunlan을 인정합니다. 이 연구는 로버트 A. 웰치 재단 (A - 1585)에 의해 부분적으로 지원되었다.

시약의 준비 파이프 버퍼 (코와 산도 6.8로 조정 80 MM 파이프, 1mM MgCl 2, 1mM EGTA) 준비. -70에서 나누어지는 및 매장 포도당 산화 효소, 카탈 라 아제 및 포도당 -20 ° C에서와 taxol ° C 기존의 프로토콜에 따라. -70에서 1,2 나누어지는 및 매장 tubulin ° C 공급자에 포함된 지시에 따라. 리소그래피를 사용하여 패턴 오 microelectrodes <ol><li> 학자 또는 웨이퍼 커터를 사용하여 1cm &#39;1.5 cm 기판으로 실리콘 웨이퍼를 자르고. 그들을 커버하고 5 분 sonicate 정도 아세톤있는 비커에서 그들을 배치하여 기판을 청소하십시오. 기판 건조 수 있도록하지 않고, 신선한 아세톤에서 그들을 씻어 후 즉시 이소프로판올 건조 질소로 사용 린스. </li><li> 원하는 기능의 크기에 따라, 표준, 긍정적인 톤 석판술이나 전자빔 리소그래피를 사용하여 깨끗한 기판에 오 microelectrodes를 조작. 3 패턴 지오메 트리를 디자인할 때,의 범위를 보장하기 위해 100 미크론 아래의 각 전극 패턴의 직경을 유지 PEG SAM (다음 섹션에서 설명). 전극 (그림 1)과 패드를 연결하는 1mm 라인 패턴에서 500 mm까지 300 위치 연락처 패드 (~ 300mm 2) 포함합니다. 흐름 챔버를 조립을위한 패턴 주위에 공간을 허용하도록 기판의 중앙 근처에 리소그래피 패턴을 놓으십시오. <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="그림 1" /> 그림 1. 샘플 기하학. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1390/1390fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다. </li><li> 저항 층의 패턴을 개발 후, 접촉 패드에서 핀셋을 (그림 1)를 사용하여 기판의 가장자리에 저항의 라인을 긁어. 금속 증착시, 스크래치는 전극과 기판의 가장자리 사이의 전기적 접촉을하실 수 있습니다. 선호 두 군데로 증착 챔버는, 금속 레이어를 입금하는 데 사용됩니다. 전극은 진공을 깨지 않고, 오 6 NM 다음 피크 2 NM의 증착 (유착)을 형성하고 있습니다. 챔버 내부에 장착되어 쿼츠 크리스털 microbalance는 증착 동안 필름의 두께를 측정하는 데 사용할 수 있습니다. </li></ol> SAM 준비 <ol><li> 앞서 열을 가하다과 75 환기가 잘되는 지역에 오븐 ° C. 250 ML 유리 비커에 톨루엔 20 ML을 추가하여 silanization 솔루션을 준비합니다. 그런 다음 플라스틱 피펫을 사용하여 톨루엔 1 ML PEG - 실란 (예 : 5% V / V)를 추가합니다. 최대 pipetting 아래 5-10 회 후 30 초 동안 피펫과 감동하여 PEG - 실란 우물에 섞는다. </li><li> 그들이 완전히 PEG - 실란 톨루엔 용액에 빠져들하고 균일하게 비커의 바닥에 걸쳐 간격 있는지 확인하고, 위쪽으로 직면하고있는 패턴 사이드와 비커의 패턴 샘플을 넣으십시오. 오븐에 비커를 놓고 75 18-21시간를 구워 ° C. SAM이 형성되는 동안 counterelectrode 준비 (아래 참조)과 나머지 단계에 필요한 추가 구성 요소를 조립. </li><li> 오븐에 18-21시간 후, 톨루엔은 점성 PEG - 실란 잔여물의 패턴 샘플을 떠나 증발한다. 비커에 톨루엔 20 ML을 추가하고 1~2분에 대한 샘플을 만끽해보세요. 비커에서 샘플을 제거한 다음 톨루엔, 이소프로판올와 린스, 그리고 질소로 드디어 건조. 이 시점에서, 샘플은 깨끗하고 보여야하고 SAM 계층은 육안으로 보이지 않는해야합니다. 샘스 시원하고 건조 조건 (25 ° C, 적당한 습도) 아래에 저장하면 최대 두 일 동안 사용할 수있는이 방법을 준비했습니다. 표면에 흐린 또는 고르지 hued 잔류 코팅에 실패한 SAM 형성 결과. </li></ol> counterelectrode를 조작 microelectrode 증착에 사용되는 동일한 증착 챔버 설정에서, 22 X 50mm 번호 1 번호 0 유리 coverslip에 오 4 NM 다음 피크 1 나노미터를 입금하여 counterelectrode합니다. counterelectrode는 밝은 회색 또는 분홍색 색조와 거의 투명해야합니다. 깨끗하게 유지하기는 배양 접시에 코팅 쪽 접속 counterelectrode를 저장합니다. MTS로 중합 tubulin <ol><li> 해동은 tubulin과 rhodamine tubulin의를 레이블이 지정되지 않은 즉시 밝은 MTS를 얻기 위해 레이블이 지정되지 않은 위해 레이블 1시 2분의 비율로 결​​합되어 있습니다. 몇 시간을 아래와 pipetting하여 섞는다. 37 온수 물을 욕조에 20~40분에 대한 tubulin을 중합 ° C. MTS의 길이는 더 이상 중합 시간에 대한 더 많은 것입니다. tubulin은 polymerizing이지만, 파이프 버퍼를 포함하는 microcentrifuge 관으로 20 mm까지 taxol을 추가하여 파이프 - taxol 버퍼를 준비합니다. 최대 pipetting 아래로 여러 번하고 vortexing하여 잘 taxol를 섞어 다음 37 온수 욕조에 튜브를 삽입 ° C. 중합 후, 따뜻한 파이프 - taxol 버퍼와 방패의 1 / 100 MTS를 희석빛과. 이런 방식으로 준비 MTS는 시간과 추가로 희석하여 줄일 수 번들을 통해 길어지다 것입니다. </li><li> 30 ML 차가운 파이프 버퍼 (0-4 ° C), 2 - 메르 캅 토 에탄올 50 %, 카탈 라 아제 5 ML 포도당 산화 효소, 5 ML, 5 ML 포도당의 5 ML을 결합하여 10X 산소 청소부 혼합 (OS)를 준비합니다. 2 혈당 마지막으로 추가되어야합니다. 각 구성 요소를 추가한 후 3-5 시간을 pipetting 아래로 섞는다. 얼음에서 운영 체제 혼합 장소, 그것은 약 2 시간 동안 효과가 유지됩니다. </li><li> MT 중합가 성공과 MTS는 영상 1X OS 믹스와 함께 준비 MTS에 의해 형광 여기의 몇 분 이내에 안정되는 것을 확인합니다. 이미징에 대해이 방법으로 준비 MTS는, rhodamine의 형광에 적합한 자극 / 방출 필터 조합을 사용해야합니다. 추가 1X OS 믹스와 파이프 버퍼와 이미징을 사용하여 10분의 1 1 / 100 MTS를 diluting하여 이미징 개별 필라멘트에 가장 적합한 작업 희석를 결정합니다. </li><li> 양면 테이프 두 가지가 떨어져 2-3밀리미터를 배치와 함께 대형 (22 X 50mm)과 작은 (22 X 22mm) 유리 coverslip을 sandwiching하여 흐름 챔버를 준비합니다. 테이프는 쉽게 유리 슬라이드에 배치하고 면도날로 절단하여 스트립으로 절단됩니다. micropipette를 사용하여 흐름 세포에 MTS 몇 microliters을 소개합니다. 높은 배율 이미징 들어, 긴 작동 거리의 목표는 이미징 흐름 챔버의 위쪽 표면을 위해 필요할 수 있습니다. 흐름 챔버의 상단 표면에 coverslip / 샘플 표면에서 거리는 테이프의 두께에 의해 결정되며 60~1백밀리미터해야합니다. </li></ol> 패턴 MTS는 전기를 사용하는 <ol><li> 전극이 테이프 (그림 2) 수직 실행 연결 경로로 스트립 사이의 이러한 패턴, SAM - 코팅 샘플에 걸쳐 양면 테이프 두 얇은 스트립을 삽입하여 전기 챔버를 준비합니다. 같은 테이프를 감동 금속 면을 양면 테이프에 counterelectrode를 놓고 그것에 대해 3mm (그림 2)에 의해 샘플 overhangs counterelectrode의 가장자리. 절연 구리 마그넷 와이어의 여러 15cm 길이를 잘라 양쪽의 절연의 1cm를 스트립. counterelectrode (이것은 멀티미터로 확인하실 수 있습니다)와 전기 접촉을 만드는 않도록주의하면서 실버 페인트의 작은 방울을 사용하여 기판의 노출된 오 영역에 철사를 연결합니다. 은색 페인트를 사용하여 counterelectrode에 두 번째 와이어를 연결합니다. 은색 페인트와 연결된 전선은 더욱 테이프와 함께 샘플을 확보할 수 있습니다. <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="그림 2" /> 거꾸로 형광 현미경을위한 그림 2. 플로우 셀 조립. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1390/1390fig2.jpg">여기를 클릭</a> 그림 2의 더 큰 버전을 볼 수 있습니다. </li><li> 1X 최종 농도에서 OS와 함께 파이프 버퍼를 사용하여 원하는 희석에서 MTS를 소개합니다. 20X 확대 이미지와 전압을 적용하기 전에 실리콘 웨이퍼에 패턴을 찾습니다. 선택, 흐름 챔버는 흐름 챔버 비행기에서 유체 흐름과 MT 마이 그 레이션을 방지하기 위해 손톱 폴란드 사용하여 봉인 수 있습니다. </li><li> 최대 1 V로 전압을 적용하고 MTS의 마이 그 레이션을 관찰합니다. MTS의 가역 트래핑이 원하는 경우 낮은 전압 (아래 0.5 V로)을 사용할 수 있지만 이것은 측면 표류을 방지하기 위해 흐름 챔버를 씰링이 필요합니다. 매니큐어의 흐름 챔버의 열린 끝을 밀봉을 위해 잘 작동합니다. 0.7 V 이상의 전압에 대해에서 일부 MT 부착이 그것이 약한 것입니다 불구하고, 패턴을 볼 수 있습니다. 0.9 V 이상의 전압에 관한에서는 MT 마이 그 레이션이 빠르게되고, 접착력이 강한 것입니다. 약 1.2 V 위의 microelectrodes을 파괴 또한 유도 전기 (가스 기포 형성)과에 대한 가능성이 크게 높아집니다. 마이 그 레이션은 현미경의 초점의 수직 비행기를 조정하여 추적할 수 있습니다. 정확하게 수행하면 MTS는 전극 표면에 집중합니다. </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.

MTS의 마이 그 레이션을 쉽게하기 때문에 그들의 밝기와 낮은 배경 형광의 형광 현미경을 사용하여 시각입니다. 그것은 biomolecules이 버퍼 산도의 적절한 조정하여 그물 요금을 휴대 유도해야합니다와 같은 방법은 일반적으로 생분자 patterning 적용됩니다. 이러한 모세관 전기 영동에 사용되는 실리카 벽으로 더 충전 표면이 없기 때문에 Electroosmosis이 설정에서 피할 수있다. 이 ?...

제 1 borosilicate 유리 coverslips (22 X 50mm)를 VWR에서 구입한했다. 광택 전자 학년 P - 타입 &lt;111&gt; 실리콘 웨이퍼는 고순도 실버 페인트가 구조 프로브, 주식 회사 (카탈로그 # 5001 AB)로부터 얻은 것입니다 애디슨 엔지니어링 주식 회사에서 구입한했다. 절연 좋은 게이지 구리 마그넷 와이어는 (~ 0.08 mm (0.003) 직경) 전자 공급 업체에서 구할 수 있습니다.

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

우리는 금을 microelectrodes과 실리콘 기판에 폴리 (에틸렌 글리콜) (PEG) trimethoxysilane 자기 조립 monolayer (SAM)을 형성을위한 절차를 제시한다. PEG - SAM은 단일 어셈블리 단계에서 형성과 실리콘과 금색 표면에 biofouling을 방지합니다. SAM은 표준 긍정적인 톤 리소그래피로 패턴 외투 microelectrodes하는 데 사용됩니다. 예를 들어 미세 소관을 사용하여, 우리는 되돌릴 방법으로 SAM - 코팅 전극에 전기 마이 그 레이션을 유도하는 DC 전압을 적용합니다. 흐름 챔버은 이미징을위한 전기 마이 그 레이션 및 미세 소관의 patterning을 사용합니다<em&gt; 현장에서</em&gt; epifluorescence 현미경을 사용하여. 그것이 목표 분자를 무력화하고 따라서 특정 분자 상호 작용을 요구하지 않는 전기를 고용으로이 방법은 일반적으로 생분자 patterning 적용됩니다. 또한, 그것은 패턴에 SAM 분자 직접 리소그래피 기술을 사용하여 시도할 때 발생 문제를 방지합니다. 전자 빔 리소그래피와 호환성이 메서드는 nanoscale에서 패턴 biomolecules하는 데 사용할 수 있습니다.

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

우리는 황금 microelectrodes와 실리콘 기판에 폴리 (에틸렌 글리콜) 자기 조립 monolayer을 (PEG - SAM) 형성을위한 절차를 제시한다. PEG - SAM은 단일 단계에서 형성과 실리콘과 금색 표면에 biofouling을 방지합니다. 전기 영동은 다음 nanoscale patterning의 biomolecules에 대한 아래 사용되고 있습니다.

Patterning의 Biomolecules에 대한 Microelectrodes에 자기 조립 Monolayers을 Antifouling

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

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JoVE Journal

Biology

11.4K 조회수.  Texas A&M University (TAMU). 우리는 황금 microelectrodes와 실리콘 기판에 폴리 (에틸렌 글리콜) 자기 조립 monolayer을 (PEG - SAM) 형성을위한 절차를 제시한다. PEG - SAM은 단일 단계에서 형성과 실리콘과 금색 표면에 biofouling을 방지합니다. 전기 영동은 다음 nanoscale patterning의 biomolecules에 대한 아래 사용되고 있습니다.

비디오: 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.

광학 투명 인듐 틴 산화물 전극에 Patterning 셀

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

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생물학

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.

바이오 플립 칩을 사용하여 배아 줄기 세포의 Patterning

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.

표면 고정화 Biomolecules의 단일 분자 형광 측정을위한 자동화된 시스템

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.

성인 마우스 Cardiomyocytes의 단세포 Transcriptional 프로파일

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.

단백질 지혜 : 용 워크 벤치<em&gt; 실리코에</em&gt;<em&gt; 드 노보</em생체 분자의&gt; 디자인

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.

일렉트로을 통해 생활 미생물의 국제화 및 형광 생체 분자의 관측

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.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

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.

특정 게놈 지역과 enChIP으로 관련 분자의 식별의 분리

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.

사이트별 단백질 Lysine Acetylation 및 Succinylation 산출할 데이터 독립적인 수집 질량 분석을 사용 하 여 정량화

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.

SA-β-갈락토시다아제 계 선별 분석법

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 ...