Name: highly sensitive and rapid fluorescence detection with a portable fret analyzer
Uploaded: 2016-10-01T13:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer at JoVE.com

이 연구는 글로벌 프론티어 사업 (2011-0031944)과 KRIBB 연구 이니셔티브 프로그램의 지능형 합성 생물학 센터에서 보조금에 의해 지원되었다.

바이오 센서 1. 준비 <ol><li> 이전에 확립 된 프로토콜 2에 따라 CFP-MBP-YFP-을 His6 - 플라스미드 pET21a (+)를 구축합니다. </li><li> 대장균 DE3 균주의 단일 콜로니로 루리아 국물 (LB) 5 ㎖를 접종하고 진탕 16 시간 동안 37 ° C에서 품어. </li><li> 전송 100 ㎖ LB를 함유하는 600 나노 미터 (OD 600)에서의 광학 밀도가 0.5 (약 3 시간)에 도달 할 때까지 진탕 배양기에서 37 ℃에서 부화 500 mL의 플라스크에 O / N 배양 1 ㎖. </li><li> 4 ℃에서 20 분 동안 1000 × g에서 원심 분리하여 50ml의 원뿔형 튜브에 세포를 수확. </li><li> 4 ℃에서 20 분 동안 1,000 × g에서 50 ml의 얼음처럼 차가운 증류수 (DW) 및 원심 각 튜브에 신속하게 펠렛을 재현 탁. </li><li> 부드럽게 용액 (electrocompetent 세포)까지 소용돌이에 의해 10 % (v / v) 글리세롤과 빙냉 DW 50 μL에 펠렛을 재현 탁 것은 OD에 도달 (600) </suB&gt; (100). </li><li> 일렉트로 장치와 electroporate에 빙냉 전기 천공 큐벳에 CFP-MBP-YFP-을 His6 - 플라스미드 pET21a (+) 10 ng의 (AN OD 100 (600)에서 세포의 50 μL) electrocompetent 세포의 혼합물을 놓고 혼합물 (18 kV로 / ㎝, 25 μF). </li><li> 신속 완만 15 mL의 둥근 바닥 튜브에 진탕하면서 1 시간 동안 37 ℃에서 회복 하였다 부드럽게 세포를 큐벳에 1 mL의 SOC 배지를 추가하고 재현 탁. </li><li> 100 μg의 / ㎖의 앰피 실린을 함유하는 LB 플레이트 상에 세포를 확산하고 12 시간 동안 37 ℃에서 배양한다. </li><li> 루프를 사용하여 단일 콜로니를 분리 및 LB는 12 시간 동안 진탕 기에서 37 ℃에서 100 ㎍ / ml 암피실린 10ml에 콜로니를 접종. </li><li> 100 ㎍ / ml 암피실린 LB 500 ml의 종자 배양 5 mL를 첨가하고 37 ° C 진탕 배양기에서 배양 물을 배양한다. </li><li> 0.5 mM의 이소 프로필 β-D-티오 갈 락토 시드 (IPTG) 때 OD 600에 도달 추가0.5 24 시간 동안 37 ° C 진탕 배양기에서 문화를 품어. </li><li> 원심 분리기 20 분 (4 ° C를) 조심스럽게 뜨는을 제거하기위한 4,500 × g에서 세포. </li><li> 5 mL의 결합 완충액 (20 mM 트리스 - 염산, 산도 8.0, 1 ㎜ PMSF, 0.5 mM의 EDTA, 1 mM의 DTT)의 펠렛을 재현 탁. </li><li> 각각의 냉각 10 초 버스트 다음, 200 ~ 300 W에서 여섯 10 초 버스트 얼음에 세포를 초음파 처리. </li><li> 10,000 ×에서 해물을 원심 분리기 4 ° C에서 30 분 동안 g는 세포 파편 펠렛합니다. 새 컬렉션 튜브에 뜨는 (가용성 단백질)을 전송합니다. </li><li> 나 Ni-NTA 친 화성 컬럼 (5 ㎖의 부피) 상에 FRET 센서 단백질의 친 화성 정화, 부하 클리어 세포 용 해물의 4 ml의를 달성하고 빠른 단백질 액체 크로마토 그래피 (FPLC) (18)를 사용하여 크로마토 그래피 분석을 수행 할 수 있습니다. </li><li> 세척 완충액 다섯 컬럼 부피로 한번 씻어 열 I (50 mM의 포스페이트 완충액, 300 mM 염화나트륨, 10 mM의 이미 다졸, pH를 7.0). </li><li> 세척 완충액 II 다섯 컬럼 부피 (50 mM의 포스페이트 완충액, 300 mM 염화나트륨, 20 mM의 이미 다졸, pH를 7.0)으로 세척하는 단계를 반복한다. </li><li> 용출 완충액의 다섯 컬럼 부피 (50 mM의 포스페이트 완충액, 300 mM 염화나트륨 500 mM의 이미 다졸, pH를 7.0)와 센서 단백질을 용출시켰다. </li><li> 집중 용출 샘플을 탈염, 3,000 × g에서 10 분 동안 샘플을 원심 분리기의 최대 20 ㎖에 집중 (10,000 MW의 막 크기)를 입력합니다. 0.8 % 인산 완충 식염수 (PBS)로 집중 리필. 첫 번째 샘플의 20 ml의 집중을 충전 한 후 PBS로 채우는 두 번이 단계를 반복합니다. </li><li> 농축 및 탈 소금에 절인 센서 단백질 복구 및 -80 ° C에서 보관합니다. </li></ol> 프렛 분석기를 사용하여 당도 2. 측정 참고 : FRET 분석기 건설의 세부 사항은 이전 작품 7에 설명 하였다. <ol><li> 0.8 % 0.2 PBS를 함유하는 검출 솔루션을 준비센서 단백질 μM. </li><li> 프렛 분석기를 켭니다. 최적의 온도를 보정하기 위해 2 초 동안 &quot;UP&quot;버튼을 누릅니다. 은 &quot;UP&quot;과 &quot;DOWN&quot;버튼을 사용하여 53 ° C로 온도를 설정하고 &quot;SET&quot;버튼을 누릅니다. </li><li> 교정, 언론과 2 초 동안 동시에 &quot;UP&quot;과 &quot;DOWN&quot;버튼을 길게하십시오. LED가 패널에 &quot;CALIB&quot;하고는 &quot;SET&quot;버튼을 눌러 확인합니다. </li><li> 만 PBS는 분석기의 큐벳 홀더에 버퍼 및 &quot;SET&quot;버튼을 누르면이 포함 된 12.5 × 12.5 × 45mm (길이 × 폭 × 높이) 직육면체 용기 (큐벳)를 놓습니다. </li><li> 하나는 설탕 (맥아당 / 크로스)없이 오직 탐지 솔루션 (2.1 참조) 함유 큐벳을 교체하고베이스 라인을 교정하기 위해 &quot;SET&quot;버튼을 누릅니다. </li><li> 10 mM의 설탕 검출 솔루션을 포함하는 하나 큐벳을 교체하고 &quot;SET&quot;를 눌러단추. </li><li> 음료 샘플의 당 함량을 결정하기 위해 1 분 동안 16,000 × g에서 1.5 ml의 마이크로 원심 분리기 튜브에 1 ㎖의 음료 샘플을 넣어. 주 : FRET 센서 계 형광 측정 총량에 포함되는 샘플의 때문에 1 % (v / v)로 시료의 특별한 전처리를 필요로하지 않는 장점을 갖는다. 그러나, 우리는 형광 측정 (예를 들면, 세포, 불용성 입자, 지질, 지방, 또는 자기 형광과 자료를) 영향을 줄 수있는 물질을 제거하는 것이 좋습니다. 강산, 강염기, 제 (세정제)를 청소하거나, 유화제 (유화제)이 고농도로 존재하고 FRET 생물학적 센서의 특성에 영향을 미칠 수있는 경우 또한, 그 유기 용매 또는를 사용하여 제거되어야 중화제. 우유 지방, 유화제가 스낵 냉동로부터 제거되는 경우, 예를 들어, 샘플은 30 분, 액체 betwee 16,000 × g에서 미세 원심 분리 튜브에서 원심 분리N 바닥 침전물과 우유 지방의 상층을 추출한다. 헥산의 동일한 양을 30 분 지질을 제거하기 위해 15,000 × g에서 원심 분리 후 첨가한다. </li><li> 1 ML의 주사기로 상층 액을 제거하고 주사기 필터 (기공 크기 0.2 μm의)를 통해 필터링합니다. </li><li> 부드럽게 0.9 ml의 PBS와 소용돌이를 포함하는 1.5 ml의 microcentrifuge 관에서 음료 샘플 필터링 0.1 ml에 놓습니다. 참고 : 제대로 음료 샘플을 희석하는 것이 중요합니다. 당 농도가 상기 장치의 동적 범위에 속하는 것이라고이 경우, 1000 배 희석을 수행 하였다. 우리는 음료의 라벨의 당도를 참조하여, 사전에 목표 당 농도를 추정 바랍니다. </li><li> 검출 용액 0.495 mL를 함유하는 큐벳 (1 % v / v)로 희석 된 음료 시료 5 μL를 추가한다. </li><li> 프렛 분석기의 큐벳 홀더에 큐벳을 놓고 53 ° C에 시료 용액을 예열. </li><li> 설탕 함량을 측정 할 수있는 &quot;SET&quot;버튼을 누릅니다. 535분의 488 내지 7,8의 비율을 판독하여 온도 제어하는 펠티에 소자를 구비 한 다중 레벨 플레이트 판독기 또는 형광 분광 광도계를 이용하여 FRET 측정을 평가하는 것이 가능합니다. 크로스 탐지를 들어, CSY-LH 센서 2 2.12 1.1의 단계를 따릅니다. </li></ol>

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저자는 공개 아무것도 없어.

이 프로토콜은 FRET 센서 최적 온도 주문품 FRET 분석 장치 (7)를 이용하여 음료 샘플 당 농도의 신속하고 효율적인 정량화를 허용한다. 이 분석기는 최근에 개발 된 저렴한 405 나노 밴드 광원 및 실리콘 포토 다이오드와 두 개의 광 검출기로 자외선이-LED 설계되었습니다. 이 장치는보다 비용 효율적인 다른 비교 fluorometers보다. FRET 센서를위한 최적의 온도 범위에서 두 개의 발광 파장 대역 (53...

포스터 공명 에너지 전달 (FRET)을 널리 당, 칼슘 이온, 아미노산 1-4와 같은 작은 분자를 검출하는 바이오 센서로서 사용되어왔다. FRET 바이오 센서는 형광 단백질, 시안 형광 단백질 (CFP들), 및 주변 세포질 결합 단백질 (PBPs)의 양단에 융합 노란색 형광 단백질 (YFPs)를 포함한다. 당류는 이후 PBPs 양쪽 끝에 두 형광 단백질의 거리 및 전이 쌍극자의 방향을 변경하는 센서의 구조 변화를 일으키는 FRET 센서의 중앙에 위치 PBPs 바인딩. 이러한 변화는 EYFP (530 ㎚) 및 ECFP (480 ㎚)의 발광 파장의 비율을 측정함으로써, 당 함량의 정량 분석을 가능하게한다. 높은 민감도, 특이도, 실시간 모니터링 능력 및 FRET 바이오 센서의 빠른 응답 시간으로 인해, 이러한 센서는 크게, 환경 산업, 의료 분야 5에 사용된다. 또한, ratiom이 센서의 농도를 용이하게 제어 및 배경 형광이 항상 존재할 수없는 복잡한 생물학적 시료의 성분을 측정하기 위해 사용될 수있다 FRET 바이오 센서를 사용하여 측정 etric 중요한 실용적인 장점을 갖는다. 양적 시각화를위한 FRET 기반 센서의 이러한 장점에도 불구하고, 형광 단백질에 불완전 도메인 모션 전송 작은 구조적 변화는 본질적으로 약한 신호 강도를 생산하고 있습니다. 이 약한 신호는 시험 관내 또는 생체 내 분석 6 FRET 기반 센서의 적용을 제한합니다. 따라서, 대부분의 바이오 센서가 고가 민감한 장비를 사용해야 FRET. 이전에는, 기존 형광 분석기 (7)의 것과 유사한 기능을 저렴하고 휴대용 FRET 분석을 개발 하였다. 이 장치 저렴 405 nm의 대역 자외선 발광 다이오드 (LED)에서는 제의 여기를 야기하는 광원으로서 사용 된즉 형광 신호 고가 램프 또는 레이저를 교체. 광자의 검출 시스템은 효율적으로 실리콘 광 다이오드 두 광 검출기 상으로 발산 형광 신호를 집중한다. 55 ° C를 크게 비율 계량 FRET 신호 (8)을 확대 할 수 - 더 최근의 연구에서 우리는 50에서 검출 온도의 최적화는 것을 보여 주었다. 이 온도 별 신호 향상은 맞춤형 FRET 분석과 함께 신속한 고감도 일반적인 진단 애플리케이션 FRET 센서의 사용을 가능하게한다. 이 프로토콜에서는 시판 음료의 당 함량을 정량함으로써 FRET 최적 온도 조건 하에서 분석 FRET의 일반적인 적용 가능성을 보여 주었다. 이 프로토콜은 FRET 장치 동작의 세부 사항뿐만 아니라 센서 샘플 준비에 대한 간략한 설명을 제공한다. 우리는이 보고서는 휴대용의 잠재적 인 응용 프로그램을 촉진 것으로 예상소규모 실험실 환경 분석기 FRET 기반 바이오 센서와 저가의 현장 진단 장치의 추가 개발을위한 기반을 제공한다.

<table><tbody><tr><td>LB</td><td>BD</td><td>#244620</td><td></td></tr><tr><td>isopropyl &amp;beta;-D-thiogalactoside (IPTG)</td><td>Sigma</td><td>I6758</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>Tri-HCl</td><td>Bioneer</td><td>C-9006-1</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>78830</td><td></td></tr><tr><td>EDTA</td><td>Bioneer</td><td>C-9007</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>NaCl</td><td>Junsei</td><td>19015-0350</td><td></td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Gibco</td><td>70011-044</td><td>0.8% NaCl, 0.02% KCl, 0.0144% Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.024% KH&lt;sub&gt;2&lt;/sub&gt;OP&lt;sub&gt;4, &lt;/sub&gt;pH 7.4</td></tr><tr><td>SOC</td><td></td><td></td><td>2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl&lt;sub&gt;2&lt;/sub&gt;, 20 mM Glucose</td></tr><tr><td>Resource Q</td><td>Amersham Biosciences</td><td>17-1177-01</td><td>6 &amp;times; 30 mm anion-exchange chromatography column&amp;nbsp;</td></tr><tr><td>HisTrap HP1</td><td>Amersham Biosciences</td><td>29-0510-21</td><td></td></tr><tr><td>Quartz cuvette</td><td>Sigma</td><td>Z802875</td><td></td></tr><tr><td>AK&amp;Auml;KTAFPLC</td><td>Amersham Biosciences</td><td>18-1900-26</td><td>a fast protein liquid chromatography (FPLC)</td></tr><tr><td>Cary Eclipse</td><td>VarianInc</td><td></td><td>a fluorescence spectrophotometer</td></tr><tr><td>VICTOR&amp;nbsp;&amp;nbsp;</td><td>PerkinElmer</td><td>2030-0050</td><td>a multilabel plate reader</td></tr><tr><td>&lt;i&gt;E. coli&lt;/i&gt; JM109 (DE3)</td><td>Promega</td><td></td><td>Electrocompetent cells</td></tr><tr><td>A (Beverage)</td><td>Korea Yakult Co. (Korea)</td><td>Birak</td><td>Fermented drinks</td></tr><tr><td>B (Beverage)</td><td>Lotte Foods (Korea)</td><td>Epro</td><td>Soft drink</td></tr><tr><td>C (Beverage)</td><td>Lotte Foods (Korea)</td><td>Getoray</td><td>Sports drink</td></tr></tbody></table>

highly sensitive and rapid fluorescence detection with a portable fret analyzer

포스터 공명 에너지 전달이 최근 개선 (FRET) 센서는 이온 및 아미노산을 포함하여 다양한 작은 분자를 검출하기 위해 사용을 사용할 수있다. 그러나, FRET 센서의 본래 약한 신호 세기는 다양한 분야에의 응용을 방지하고 비싼 고성능의 사용이 필요하게 fluorometers 중요한 과제이다. 이전에, 우리는 특히 높은 검출 감도를 달성하기 위해 두 개의 발광 파장 대역 (530 및 480 ㎚)의 비율을 측정 할 수있는 경제적 인 고성능 FRET 분석 내장. 55 °의 C - 더 최근에는 세균의 세포질 단백질과 결합 FRET 센서 (50)의 임계 온도 범위에서 최대 감도 리간드를 검출하는 것이 발견되었다. 이 보고서는 온도 별 FRET 센서 우리 FRET 휴대용 분석기를 사용하여 시판 음료 샘플 당 함량을 평가하기위한 프로토콜을 설명한다. 우리의 결과는 추가 예열 것으로 나타났다프렛 센서의 과정은 상당히 당도의보다 정확한 측정을 가능하게하려면 FRET 비율 신호를 증가시킨다. 주문품 FRET 분석과 센서 성공적 상용 음료의 세 종류의 당 함량을 정량하기 위해 적용 하였다. 우리는 하이 엔드 장비는 가능하지 않은 장치의 추가적인 소형화 및 성능 향상이 환경에서 휴대용 분석기의 사용을 용이하게 할 것으로 예상된다.

프렛 분석기를 사용 당도의 정량 분석을 수행하기 위해, 관찰 된 FRET 비율에서 표적 당 농도를 추정 피팅 곡선을 구축 할 필요가있다. R은 480 nm에서 CFP의 발광 강도의 비율을 정의 YFP의 발광 강도가 530 나노 미터 (식. 1)에서 생성하자. <img alt="식 (1)" src="/files/ftp_upload/54144/54144equation1.jpg" /><p class="jove_content" fo:keep-together....

Watch this Scientific Journal Video about Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer at JoVE.com

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

이 프로토콜은 주문품 휴대용 FRET 분석기를 사용하여 포스터 공명 에너지 전달 (FRET) 센서 데이터의 신속한 고감도 정량 설명한다. 장치는 당도 실용적이고 효율적인 평가를 가능하게 검출 감도를 최대화하는 임계 온도 범위 내에서 말토오스를 검출하기 위해 사용 하였다.

휴대용 FRET 분석과 고감도 및 고속 형광 검출

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

highly-sensitive-rapid-fluorescence-detection-with-portable-fret

Research

JoVE Journal

Biochemistry

9.0K 조회수.  Korea Research Institute of Bioscience and Biotechnology.  이 프로토콜은 주문품 휴대용 FRET 분석기를 사용하여 포스터 공명 에너지 전달 (FRET) 센서 데이터의 신속한 고감도 정량 설명한다. 장치는 당도 실용적이고 효율적인 평가를 가능하게 검출 감도를 최대화하는 임계 온도 범위 내에서 말토오스를 검출하기 위해 사용 하였다. 

비디오: Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

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, ...

연구

생물학

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

살아있는 세포 형광 기술의 조합을 사용 하 여 힘에 민감한 단백질 역학의 측정

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

생체공학

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

안달 Ratiometric하여 세포주기를 통해 키나제와 인산 가수 분해 효소 활동을 모니터링

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

Unimolecular 바이오 센서를 사용하여 라이브 세포에서 이벤트를 신호의 실시간 모니터링을위한 현미경을 안달

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

모바일 단일 분자 FRET 프로브의 자동화된 2차원 스파티오 측량 분석

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

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 ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

3 차원 히드로 겔의 이미지를 FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

자동 형광 수명 이미징 현미경을 활용하여 오픈 소스 높은 콘텐츠 분석

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

라이브 셀의 형성 FRET 바이오 센서를 사용하여 오로라 키나아제 활성화의 실시간 모니터링

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

FRET-Sensitized Emission with FRET-Sensitized Release를 사용한 살아있는 세포의 단백질 상호 작용 평가

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...