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>

<ol>
	<li>Deuschle, K., Okumoto, S., Fehr, M., Looger, L. L., Kozhukh, L., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+optimization+of+a+family+of+genetically+encoded+metabolite+sensors+by+semirational+protein+engineering.">Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering.</a> Protein Sci. 14 (9), 2304-2314 (2005).</li><li>Ha, J. S., Song, J. J., Lee, Y. M., Kim, S. J., Sohn, J. H., Shin, C. S., Lee, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+application+of+highly+responsive+fluorescence+resonance+energy+transfer+biosensors+for+detection+of+sugar+in+living+Saccharomyces+cerevisiae+cells.">Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells.</a> Appl. Environ. Microbiol. 73 (22), 7408-7414 (2007).</li><li>Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+dynamic+range+of+fluorescent+indicators+for+Ca(2+)+by+circularly+permuted+yellow+fluorescent+proteins.">Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (29), 10554-10559 (2004).</li><li>Okumoto, S., Looger, L. L., Micheva, K. D., Reimer, R. J., Smith, S. J., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+glutamate+release+from+neurons+by+genetically+encoded+surface-displayed+FRET+nanosensors.">Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (24), 8740-8745 (2005).</li><li>Merzlyakov, M., Li, E., Casas, R., Hristova, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+F&#246;rster+resonance+energy+transfer+detection+of+protein+interactions+in+surface-supported+bilayers.">Spectral F&#246;rster resonance energy transfer detection of protein interactions in surface-supported bilayers.</a> Langmuir. 22 (16), 6986-6992 (2006).</li><li>Zhang, J., Campbell, R. E., Ting, A. Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+new+fluorescent+probes+for+cell+biology.">Creating new fluorescent probes for cell biology.</a> Nat. Rev. Mol. Cell Biol. 3 (12), 906-918 (2002).</li><li>Kim, H., Kim, H. S., Ha, J. S., Lee, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+portable+FRET+analyzer+for+rapid+detection+of+sugar+content.">A portable FRET analyzer for rapid detection of sugar content.</a> Analyst. 140 (10), 3384-3389 (2015).</li><li>Gam, J., Ha, J. -. S., Kim, H., Lee, D. -. H., Lee, J., Lee, S. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+analyses+at+critical+temperatures+can+magnify+the+signal+intensity+of+FRET-based+sugar+sensors+with+periplasmic+binding+proteins.">Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins.</a> Biosens. Bioelectron. 72, 37-43 (2015).</li><li>Hessels, A. M., Merkx, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically-encoded+FRET-based+sensors+for+monitoring+Zn2++in+living+cells.">Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cells.</a> Metallomics. 7 (2), 258-266 (2015).</li><li>Song, Y., Yang, M., Wegner, S. V., Zhao, J., Zhu, R., Wu, Y., He, C., Chen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+FRET+sensor+for+intracellular+heme.">A genetically encoded FRET sensor for intracellular heme.</a> ACS Chem. Biol. 10 (7), 1610-1615 (2015).</li><li>. Fluorescent Protein Guide: Biosensors Available from: <a target="_blank" href="https://www.addgene.org/fluorescent-proteins/biosensors/">https://www.addgene.org/fluorescent-proteins/biosensors/</a> (2015)</li><li>Rajendran, R., Rayman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+blood+glucose+testing+for+diabetes+care+in+hospitalized+patients:+an+evidence-based+review.">Point-of-care blood glucose testing for diabetes care in hospitalized patients: an evidence-based review.</a> J. Diabetes Sci. Technol. 8 (6), 1081-1090 (2014).</li><li>Vyas, N. K., Vyas, M. N., Quiocho, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+and+signal-transducer+binding+sites+of+the+Escherichia+coli+galactose+chemoreceptor+protein.">Sugar and signal-transducer binding sites of the Escherichia coli galactose chemoreceptor protein.</a> Science. 242, 1290-1295 (1988).</li><li>Leermakers, E. T. M., Felix, J. F., Erler, N. S., &#266;erimagi&#263;, A., Wijtzes, A. I., Hofman, A., Raat, H., Moll, H. A., Rivadeneira, F., Jaddoe, V. W., Franco, O. H., Kiefte-de Jong, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar-containing+beverage+intake+in+toddlers+and+body+composition+up+to+age+6+years:+The+Generation+R+Study.">Sugar-containing beverage intake in toddlers and body composition up to age 6 years: The Generation R Study.</a> Eur. J. Clin. Nutr. 69 (3), 314-321 (2015).</li><li>Shilts, M., Styne, D., Drake, C., Aden, C., Townsend, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+food,+fat+and+sugar+sweetened+beverage+items+are+related+to+children's+dietary+energy+density.">Fast food, fat and sugar sweetened beverage items are related to children's dietary energy density.</a> FASEB J. 29 (1), 731-736 (2015).</li><li>Larsson, S. C., &#197;kesson, A., Wolk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweetened+beverage+consumption+is+associated+with+increased+risk+of+stroke+in+women+and+men.">Sweetened beverage consumption is associated with increased risk of stroke in women and men.</a> J Nutr. 144 (6), 856-860 (2014).</li><li>Melkko, S., Neri, D., Vaillancourt, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+as+an+affinity+purification+tag.">Calmodulin as an affinity purification tag.</a> E. coli Gene Expression Protocols. , 69-77 (2003).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1297">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">LB</td>
			<td style="width:191px;">BD</td>
			<td style="width:119px;">#244620</td>
			<td style="width:760px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">isopropyl &#946;-D-thiogalactoside (IPTG)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">I6758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ampicillin</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Tri-HCl</td>
			<td style="width:191px;">Bioneer</td>
			<td style="width:119px;">C-9006-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">PMSF</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">78830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">EDTA</td>
			<td style="width:191px;">Bioneer</td>
			<td style="width:119px;">C-9007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">DTT</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">NaCl</td>
			<td style="width:191px;">Junsei</td>
			<td style="width:119px;">19015-0350</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">phosphate-buffered saline (PBS)</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:119px;">70011-044</td>
			<td>0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SOC</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl2, 20 mM Glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Resource Q</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">17-1177-01</td>
			<td>6 &#215; 30 mm anion-exchange chromatography column&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">HisTrap HP1</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">29-0510-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Quartz cuvette</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">Z802875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">AK&#196;KTAFPLC</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">18-1900-26</td>
			<td>a fast protein liquid chromatography (FPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cary Eclipse</td>
			<td>VarianInc</td>
			<td style="width:119px;"></td>
			<td>a fluorescence spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">VICTOR&#160;&#160;</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">2030-0050</td>
			<td>a multilabel plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">E. coli JM109 (DE3)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;"></td>
			<td>Electrocompetent cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">A (Beverage)</td>
			<td style="width:191px;">Korea Yakult Co. (Korea)</td>
			<td style="width:119px;">Birak</td>
			<td>Fermented drinks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">B (Beverage)</td>
			<td style="width:191px;">Lotte Foods (Korea)</td>
			<td style="width:119px;">Epro</td>
			<td>Soft drink</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">C (Beverage)</td>
			<td style="width:191px;">Lotte Foods (Korea)</td>
			<td style="width:119px;">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

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

생체 분자 구조 결정을위한 단일 분자 수준의 고정밀 FRET

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

연구

생화학

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.

This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.

실시간 세포 분석기 장착 기준점과 골드 Microelectrodes의 재생

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Rab10 인 산화 탐지 LRRK2 활동 인산 염-바인딩 태그와 SDS 페이지를 사용 하 여

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

단백질 및 모 세관 전자 초점 방법으로 그들의 Isoforms의 매우 민감하고 정량적 검출

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

세제에 민감한 막 단백질 사이 상호 작용의 탐지

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

작은 GTPase Prenylation와 GTP 바인딩 사용 하 여 막 분류 및 GTPase 연결 된 Immunosorbent 분석 결과의 검색

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

나노-추적 분석 인 혈에 있는 단일 Extracellular Vesicles의 빠른 형광 기반 특성

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

펩타이드 농축 및 질량 분광법에 의한 단백질 유비퀴틴화 부위 검출

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

차에서 파생된 새로 설립된 세포 현탁액 배양에서 6개의 전신 살충제의 물질대사에 관한 연구(동백나무시넨시스 L.) 잎

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

4-티우라실(Ers4tU)을 가진 생체내 RNA의 매우 신속하고 특정한 대사 라벨링

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...