우리는 귀중한 조언 빅터 GJ 로저스 매우 감사하고 있습니다. 우리는 아주 가까운 공동 작업에이 연구에 도움 리아 그룹의 구성원 모두들 감사합니다. 이 연구는 국립 보건원 (JL에 부여 AI076504)에 의해 지원되었다.

1. 플라스미드 구조 <ol><li> PCR에 의해 유전자의 오픈 읽기 프레임을 증폭하고, PCRII-TOPO 벡터로 PCR 제품을 복제. </li><li> 시퀀싱하여 제품을 확인하고, 복제 cDNA 인코딩 CyPet - (사전 SUMO1) YPet, CyPet - SUMO1, YPet 및 N-터미널 hexahistidine 태그와 pET28 (b)는 벡터에 SENP1의 촉매 도메인입니다. </li></ol> 2. 단백질 표현과 정화 <ol><li> pET28로 변형 BL21 (DE3)의 대장균 세포를 변환 (B) CyPet - (사전 SUMO1) YPet, CyPet - SUMO1, YPet 및 SENP1의 촉매 도메인을 인코딩 벡터. </li><li> 600 나노 미터에서 0.5의 광학 밀도에 도달 할 37 ° C (250 rpm으로 흔들어)에서 3 시간에 2xYT 매체에 변형 박테리아를 성장. </li><li> 이소 프로필-β-D-thiogalactoside (IPTG) (최종 농도)은 단백질 식을 유도 25 16 시간에 흔들 ° C를 200 rpm으로하여 100 μM을 추가합니다. </li><li> centrifug하여 박테리아를 수확4에 10,000 RPM에서 ation ° 5 분, 20 MM 트리스 - HCL (산도 7.4), 50 MM NaCl, 5 MM 이미 다졸의 버퍼에서 그들을 resuspend을위한 C. </li><li> MiSonics 4000 sonicator를 사용하여 25 W의 전력 설정에서 5 초 간격으로 10 분의 세포를 Sonicate와 4에서 35,000 XG에서 원심 분리하여 수집 ° C를 30 분에. </li><li> 1 L의 문화 500 ML 니켈 NTA 비즈과 열을 설정하고 열에 표면에 뜨는을 전송합니다. </li><li> 20 MM 트리스 - HCL (산도 7.4), 500 MM NaCl, 그리고 두 번 10 MM 이미 다졸의 버퍼로 수지를 씻으십시오. </li><li> 20 MM 트리스 - HCL (산도 7.4), 500 MM NaCl, 500 MM 이미 다졸, 20 MM 트리스 - HCL (산도 7.4), 50 MM NaCl, 1 MM dithiothreitol (DTT)에서의 버퍼로 투석의 버퍼와 Elute 단백질 4 ° C 하룻밤. </li><li> 브래드 포드 분석하여 정화 단백질의 농도를 결정합니다. 단백질 농도 측정을위한 대체 방법은 SDS-PAGE 젤 전기 영동이 될 한 후 Coomassie 파란색으로 물들 예정 영상과 함께 다음양적. </li></ol> 3. 양적는 스펙트럼 분석 안달 <ol><li> 분석을위한 일반적인 전략은 (그림 1) 신호 걱정을 기반으로했다. 초조 한 쌍​​은 CyPet 및 YPet은 사전 SUMO1 때문에, 각각 N-및 C-말단에 태그되었습니다. SENP1 cleaves는 퓨전 SUMO1의 C-말단에있는 Gly-Gly 사이트에서 단백질 CyPet - (사전 SUMO1) YPet하고, 따라서 YPet와 스모 꼬리를 출시. 이 신호가 CyPet에서 방출의 증가와 CyPet 여기 파장에서 YPet의 방출의 극적인 감소의 결과로, 중단되어 걱정. </li><li> 절대 걱정 유도 YPet의 방출, CyPet 직접 방출하고 YPet 직접 배출 (그림 : 530 nm의에서 파장의 빛에 의해 414 nm 정도의 총 형광 방출 CyPet - (사전 SUMO1) YPet을 흥분 시절 세 번의 소스에서 파생 될 수 2). </li></ol><img alt="수식 1" src="/files/ftp_upload/4430/4430eq1.jpg" /> 곳 FL 414분의 530 &lt;/ 서브&gt; 414 nm의에서 흥분 530 나노 미터의 총 형광 방출는, FL는 걱정은 절대이 신호를 안달이고, FL CyPet (cont)은 414 nm의에서 흥분 CyPet 직접 방출하며, FL YPet (cont)는 YPet입니다 414 nm의에서 흥분 할 때 방출을 지시. 의 첨자 (cont)는 기부금을 의미합니다. <ol start="3"><li> α의 일정한 비율로 414 nm의에서 흥분 할 때 530 nm의에서 CyPet의 직접 방출 475 nm의에서의 방출에 비례했다. CyPet - SUMO1은 50의 농도, 100, 200, 500, 750 nm의에서 준비 1 ㎜되었습니다 , 그리고 475와 530 nm의에서 배출은 α (그림 3-1) 결정하기 위해 414 나노 미터의 여기 이후에 측정 하였다. </li><li> β의 일정한 비율로 475 nm의에서 흥분 할 때 414 nm의에서 여기에 따라 530 nm의에서 YPet의 직접 배출량은 530 nm의에서의 방출에 비례했다. YPet 50의 농도, 100, 200, 500, 750에 준비되었다샘플 β (그림 3-2)을 결정하기 위해 414의 파장과 475 nm의에 의해 흥분했을 때 차 1,000 NM 및 530 nm의에서 방출이 측정되었다. </li><li> CyPet - (사전 SUMO1) YPet은 SENP1에 의해 소화되었을 때, 절단은 CyPet - SUMO1과 YPet과 SUMO1 꼬리를 발표했다. 화합물은 414 nm의에서 흥분했을 때, 530 nm의 (FL &#39;414분의 530)에서 형광 방출이 감소되었지만 여전히 같은 세 부분으로 나눌 수 있습니다 : </li></ol><img alt="수식이" src="/files/ftp_upload/4430/4430eq2.jpg" /> FL &#39;414 nm의에서 흥분하면 414분의 530이 소화 후 530 nm의에서 총 형광 방출이며, FL&#39;어디 남은 절대 걱정 신호 걱정, FL &#39;CyPet (414분의 475)이 때 소화 후 475 nm의에서 CyPet 방출입니다 414 나노 미터 (여기서 CyPet 방출이 두 부분에서입니다 CyPet - (사전 SUMO1) YPet 소화되지 않은 및 CyPet-SUMO1을 소화)에 흥분하고, FL YPet(475분의 530)는 CyPet - (사전 SUMO1) YPet은 소화 여부를 일정 475 나노 미터에 흥분 YPet 방출입니다. <ol start="6"><li> SENP1로 소화 한 후, 나머지는 방출 (FL의 &#39;안달가)입니다 초조해 : </li></ol><img alt="수식 3" src="/files/ftp_upload/4430/4430eq3.jpg" /> C는 CyPet - (사전 SUMO1) YPet하고, x의 총 농도입니다 것은 소화의 농도 CyPet - (사전 SUMO1) YPet입니다. <ol start="7"><li> 모든 항목을 결합하여, 414 nm의 (FL &#39;414분의 530)의 흥분에 따라 530 나노 미터에서 감지 형광 방출은 다음과 같습니다 : </li></ol><img alt="수식 4" src="/files/ftp_upload/4430/4430eq4.jpg" /> 4. 효소 키네틱 연구에 대한 걱정 기반의 단백 분해 효소 분석 <ol><li> CyPet - (사전 SUMO1) YPet은 (20 MM 트리스 - HCL (산도 7.4), 50 MM NaCl, 0.1 %의 버퍼에 37 ° C에서 SENP1의 촉매 도메인 incubated되었습니다 V /V) 십대 초반-20 80 ML의 총 양이 1 ㎜의 DTT와 384도 판으로 이동. </li><li> 실행은 15 초 간격으로 5 분을위한 형광 multiwell 판 리더의 414 nm의에서 여기 이후 475과 530 nm의에서 형광 방출을 측정하여 실시되었다. </li><li> 반응 속도 (V)가 같은 기판 (S)의 양의 변화와 상관되었습니다 </li></ol><img alt="수식 5" src="/files/ftp_upload/4430/4430eq5.jpg" /><ol start="4"><li> 제품의 농도가로 0에서 기하 급수적으로 증가 [S] 0 (원래 기판 농도) 때 t = 0 : </li></ol><img alt="수식 6" src="/files/ftp_upload/4430/4430eq6.jpg" /><ol start="5"><li> t = 0은 원래 속도 (V 0) 경우 : </li></ol><img alt="수식 7" src="/files/ftp_upload/4430/4430eq7.jpg" /><ol start="6"><li> concSENP1의 entration가 해결되었습니다, 그리고 CyPet - (사전 SUMO1) YPet의 농도는 Michaelis-Menten 방정식에 필요한 효소의 농도보다 100 배 이상에서 증가되었다. </li><li> 형광 판독의 모든 정량 안달 분석 방법으로 분석하고 Michaelis-Menten 방정식에 맞게 GraphPad 프리즘 V 소프트웨어에 역모를했다. 비선형 회귀은 스프레드 시트 프로그램 (Microsoft Excel과 같은)과 같은 일반적인 소프트웨어 패키지의 도움으로 수행 할 수 있습니다. </li></ol> 5. 대표 결과 SENP1의 사전 SUMO1의 성숙이 과정에서 475과 530 nm의에서 형광 신호의 변화를 모니터링하여 결정 할 수 있습니다. 결과는 보여 그 기판 - 복용에 의존 방식으로 SENP1의 사전 SUMO1 소화의 속도 (그림 4). 이 제안 사전 SUMO1의 성숙을위한 SENP1의 촉매 도메인 전시 우수한 활동을 그. 초기 reac기의 속도는 다른 기판 농도 (표 1) 위의 분석에 의해 계산되었다. K 고양이 / K M 비율은 일반적으로 하나의 기판 또는 다른 기판과 특정 효소와 다른 효소의 효율성을 비교하는 데 사용됩니다. K M과 V 맥스 해당하는 다양한 초기 속도를, 음모에 의해 Michaelis-Menten 방정식에서 구할 수 있습니다 . CyPet - (사전 SUMO1) YPet는 (그림 5) K 고양이가로 얻은의 다른 농도로 : <img alt="수식 8" src="/files/ftp_upload/4430/4430eq8.jpg" /> 위의 분석에 따르면, 계산 된 K M은 0.21이었다 ± 0.04 μM, K의 고양이는 ± 0.28 초 -1 6.90이었고, K 고양이 / K M 비율은 (± 0.55 3.2)였습니다 X10 7 M -1 </sup<html> &gt; S -1. <img alt="그림 1" src="/files/ftp_upload/4430/4430fig1.jpg" /> 그림 1. SENP의 사전 SUMOs 성숙을위한 걱정 기반의 단백 분해 효소 분석 그래프. <img alt="그림 2" src="/files/ftp_upload/4430/4430fig2.jpg" /> 그림 2. 기증자, 수용체와 걱정의 공헌 등의 형광 신호의 정량 분석. CyPet-414 나노 미터에서 여기에 따라 (예정 SUMO1) YPet. FL CyPet (414분의 530)에서 방출 스펙트럼의 해부 414 nm의 여기에 따라 530 nm의에서 CyPet의 방출, FL가 걱정하는 걱정 유발 YPet의 414 nm의 여기에 따라 530 nm의에서 방출, 그리고 FL YPet이 (414분의 530) 414 nm의 여기에 따라 530 nm의에서 YPet의 방출이다입니다. load/4430/4430fig3.jpg &quot;/&gt; 그림 3. 방향으로 방출 계수 α와 β의 계산. <img alt="그림 4" src="/files/ftp_upload/4430/4430fig4.jpg" /> 그림 4. SENP1의 촉매 도메인의 다른 비율에 의해 소화 CyPet - (사전 SUMO1) YPet의 정량 분석. 반응은 처음 5 분 안에 모니터했다. <img alt="그림 5" src="/files/ftp_upload/4430/4430fig5.jpg" /> 그림 5. SENP1로 CyPet - (사전 SUMO1) YPet의 소화 Michaelis-Menten 그래픽 분석. 데이터는 역모와 GraphPad 프리즘 V 및 비선형 회귀하여 분석 하였다. <table border="1"><tbody><tr><td> [S] (μM) </td><td> V 0 (μM / s)의 </td></tr><tr><td> 0.115 </td><td> 0.0023 ± 0.00005 </td></tR&gt; <tr><td> 0.214 </td><td> 0.0028 ± 0.00004 </td></tr><tr><td> 0.407 </td><td> 0.0033 ± 0.00007 </td></tr><tr><td> 0.594 </td><td> 0.0037 ± 0.00013 </td></tr><tr><td> 0.725 </td><td> 0.0043 ± 0.00012 </td></tr><tr><td> 1.471 </td><td> 0.0051 ± 0.00036 </td></tr><tr><td> 1.899 </td><td> 0.0050 ± 0.00031 </td></tr><tr><td> 2.300 </td><td> 0.0050 ± 0.00062 </td></tr></tbody></table> 표 1. SENP1에서 미리 SUMO1의 성숙의 초기 속도 결정. 각 기판 농도에는, 4 샘플은 소화를 측정하기 위해 사용되었다. 표준 편차는이 네 샘플 변형에서 왔어. <table border="1"><tbody><tr><td> K M (μM) </td><td> K 고양이 (S-1) </td><td> K 고양이 / K M (M -1 • S-1) </td></tr><tr><td> 0.21 ± 0.04 </td><td> 6.90 ± 0.28 </td><td> 3.2 ± 0.55 × 10 7 </td></tr></tbody></table> 표 2. 양적으로 SENP1에서 미리 SUMO1의 성숙의 키네틱 매개 변수 분석을 초조해. 표준 편차는 각 기판 농도에 포함 된 네 개의 샘플에서 왔어.

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-resolved+fluorescence+resonance+energy+transfer-based+assay+for+DEN1+peptidase+activity.">A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity.</a> Analytical Biochemistry. 390, 85-87 (2009).</li><li>Martin, S., Hattersley, N., Samuel, I., Hay, R., Tatham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-resonance-energy-transfer-based+protease+activity+assay+and+its+use+to+monitor+paralog-specific+small+ubiquitin-like+modifier+processing.">A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing.</a> Analytical Biochemistry. 363, 83-90 (2007).</li><li>Nguyen, A. W., Daugherty, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+optimization+of+fluorescent+proteins+for+intracellular+FRET.">Evolutionary optimization of fluorescent proteins for intracellular FRET.</a> Nature Biotechnology. 23, 355-360 (2005).</li></ol>

관심 없음 충돌이 선언 없습니다.

걱정 기술은 SENP1 9 미리 SUMO1의 성숙을 연구하는 데 사용되었습니다. CFP-YFP는 기증자의 배출량에 대한 수용체의 비율입니다 걱정 쌍과 ratiometric 분석으로 사용 된 운동 속성을 특징하는 데 사용되었다. 그러나, 기존의 ratiometric의 자체 형광는 분석을 걱정 기증자와 수용체 전혀 고려가 없습니다. 비율은 직접 소화 기판의 양이 서로 관련이되지 않습니다. 여기 SENP1에 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> PCR II TOPO 키트 </td><td> Invitrogen </td><td> K466040 </td></tr><tr><td> 2xYT </td><td> 연구 제품 Internationa.l 주식회사 </td><td> X15600 </td></tr><tr><td> 니켈 NTA Agrose </td><td> 열 과학 </td><td> 88,222 </td></tr><tr><td> Coomassie 플러스 (브래드 포드) 검정 리젠트 </td><td> 열 과학 </td><td> 23,238 </td></tr><tr><td> 384 잘 플레이트 (유리 아래) </td><td> Greiner </td><td> 781892 </td></tr><tr><td> FlexStation II 384 판 리더 </td><td> 분자 장치 </td><td></td></tr></tbody></table>

quantitative fret (f&#246;rster resonance energy transfer) analysis for senp1 protease kinetics determination

ubiquitin 또는 ubiquitin 같은 단백질 (Ubls)과 단백질의 치료 posttranslational 수정은 널리 동적으로 단백질의 활동을 조절하고 다양한 생물학적 과정에서 다양한 역할을 수행하는 데 사용됩니다. 예를 들어, 스모는 covalently 세포주기 조절, 세포 생존과 죽음, DNA 손상 반응 및 스트레스 반응 1-5 등 많은 세포 과정에 중요한 역할을 갖춘 대형 번호 또는 단백질을 수정합니다. SENP는 스모 특정 단백 분해 효소로, 스모 전구체의 성숙에 endopeptidase 또는 isopeptidase의 기능은 대상 단백질에서 스모를 제거하고 SUMOylation주기에게 1,3,6,7를 새로 고칠 수 있습니다. 효소의 촉매 효율이나 특이성은 가장 운동 상수, K 고양이 / K M의 비율이 특징입니다. 여러 연구에서, 스모 - SENP 쌍의 운동 매개 변수가 힘이에요, polyacrylamide 젤 기반 서양 얼룩 등의 다양한 방법에 의해 결정되었습니다ioactive - 라벨 기판, 형광 화합물 또는 단백질이 기판 8-13를 표시. 그러나, &quot;네이티브&quot;단백질을 사용하지만 polyacrylamide - 젤 기반 기술은 힘든 및 기술적 요구이며, 그 즉시 자세한 정량 분석​​에 자신을 빌려하지 않습니다. ACC (7 - 아미노-4-carbamoylmetylcoumarin) 또는 AMC (7 - 아미노-4-methylcoumarin) 형광과 tetrapeptides 또는 단백질을 사용하여 연구에서 K 고양이 / K M을 획득 자연 기판보다 크기 낮은 최대 두 명령 중 하나였다 또는 명확하게 SENPs의 ISO와 endopeptidase 활동을 차별화 할 수 없습니다. 최근, 아가야 기반의 단백 분해 효소 assays는 시안 형광 단백질의 걱정 쌍 (CFP)와 황색 형광 단백질 (YFP) 9,10,14,15과 deubiquitinating 효소 (DUBs) 또는 SENPs을 연구하는 데 사용되었습니다. 단백 분해 효소 교류를위한 신호 모니터를 안달을 위해 기증 방출에 대한 수용체 방출의 비율은 양적 매개 변수로 사용되었다tivity 결정. 그러나,이 방법은 수용체 및 공여체 자기 형광하여 수용체와 기증자 방출 파장에서 신호 교차 contaminations을 무시하기 때문에 정확한 아니 었습니다. 우리는 SENP1에 의해 사전 SUMO1 성숙의 운동 매개 변수를 결정하는 매우 중요한 소설 및 정량 걱정 기반의 단백 분해 효소 분석을 개발했습니다. 엔지니어링은 크게 향상 걱정 효율성과 형광 양자 수율과 쌍 CyPet 및 YPet을 걱정 CyPet - (사전 SUMO1) YPet 기판 16을 생성하는 데 사용되었습니다. 우리는 차별화와 기증자와 수용체에 의해 공헌 각각의 수용체와 방출 파장에서 걱정 절대 형광 신호를 정량화. K 고양이 / K M의 값은 SENP1의 X10 7 M -1의 -1은 일반적으로 효소 운동 매개 변수 계약에 사전 SUMO1 방면 (3.2 ± 0.55)로 얻은 것입니다. 따라서이 방법은 유효하고 사용할 수 있습니다SA 일반적으로는 다른 프로테아제를 특징 접근한다.

Watch this Scientific Journal Video about Quantitative FRET FÃ¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination at JoVE.com

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

걱정의 정량 분석​​ (포스터 공명 에너지 전송) 신호를 포함하는 새로운 방법을 효소 반응 속도론을 연구에 설명되어 있습니다.<em&gt; K<sub&gt; M</sub</em&gt;와<em&gt; K<sub&gt; 고양이</sub</em&gt;은 (작은 Ubiquitin과 같은 수정) 사전 SUMO1에 SENP1 (스모 / Sentrin 특정 단백 분해 효소 1) 촉매 도메인의 가수 분해에 대한 획득했다. 이 양적 - 안달 기반 프로테아제 운동 연구의 일반적인 원리는 다른 프로테아제에 적용 할 수 있습니다.

정량은 SENP1 단백 분해 효소 반응 속도론 결정에 대해 (포스터 공명 에너지 전송) 분석 안달

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

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Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

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18.9K 조회수.  University of California, Riverside. 걱정의 정량 분석​​ (포스터 공명 에너지 전송) 신호를 포함하는 새로운 방법을 효소 반응 속도론을 연구에 설명되어 있습니다. K M

비디오: Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Micropatterned 세포를 사용하여 약물의 영향 향상된 시각화 및 정량 분석

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

연구

생체공학

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

에 대한 접착 주파수 분석 현장에서</em셀 - 셀 인터페이스에서 교차 Junctional 분자의 상호 작용> 속도론 해석

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

의 주문형 정량 분석​​을위한 미세 유체 기반 Electrotaxis<em&gt; Caenorhabditis의 엘레</em&gt; &#39;로코

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

아데 닐 레이트 사이 클라 제의 약물에 의한 과민성 : 작은 분자와 siRNA의 심사 응용 프로그램에 대한 분석 최적화 기술과 소형화

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

발광 공명 에너지는 포유 동물 세포에 표현 막 단백질의 구조 변화를 연구에 전송

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

TCR-pMHC 바인딩 측정<em&gt; 현장에서</em&gt; FRET 기반의 현미경 분석을 사용하여

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

FRET 유동 세포 계측법과 Proteopathic의 시드 활동의 민감한 검출

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

물방울 미세 유체에 대한 여러 가지 빛깔의 형광 검출 광학 섬유를 사용하여

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A Protocol for Using F&amp;#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

포스터 공명 에너지 전달 (FRET) -force의 바이오 센서를 사용하는 프로토콜은 핵 LINC 단지에서 기계의 힘을 측정하는

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

단일 벽 탄소 나노 튜브에 바이오 모방 고분자와 공학 분자 인식

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Methods Article

정량은 SENP1 단백 분해 효소 반응 속도론 결정에 대해 (포스터 공명 에너지 전송) 분석 안달