Name: in situ characterization of shewanella oneidensis mr1 biofilms by salvi and tof-sims
Uploaded: 2017-08-18T13:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS at JoVE.com

우리는 평화로운 북 서 국립 연구소 (PNNL) 지구 및 생물 과학 (EBD) 임무 씨앗 실험실 감독 연구 및 개발 (LDRD) 기금 지원에 대 한 감사. 경 음악 액세스 W. R. 윌 환경 분자 과학 실험실 (EMSL) 일반 사용자 제안을 통해 제공 되었다. EMSL은 사무실의 생물학과 환경 연구 (BER) PNNL에 주최 국가 과학적인 사용자 시설입니다. 저자는 원고를 읽고 유용한 피드백을 제공 하는 증거에 대 한 박사 Yuanzhao 딩 감사 합니다. PNNL는 Battelle 계약 드 AC05 76RL01830 아래 DOE에 대 한에 운영 됩니다.

<ol>
	<li>Renner, L. D., Weibel, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+regulation+of+biofilm+formation.">Physicochemical regulation of biofilm formation.</a> MRS Bull. 36 (5), 347-355 (2011).</li><li>Flemming, H. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat Rev Microbiol. 8 (9), 623-633 (2010).</li><li>Aldeek, F., 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=Patterned+hydrophobic+domains+in+the+exopolymer+matrix+of+Shewanella+oneidensis+MR-1+biofilms.">Patterned hydrophobic domains in the exopolymer matrix of Shewanella oneidensis MR-1 biofilms.</a> Appl Environ Microbiol. 79 (4), 1400-1402 (2013).</li><li>Hua, X., 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=In+situ+molecular+imaging+of+a+hydrated+biofilm+in+a+microfluidic+reactor+by+ToF-SIMS.">In situ molecular imaging of a hydrated biofilm in a microfluidic reactor by ToF-SIMS.</a> Analyst. 139 (7), 1609-1613 (2014).</li><li>Yu, X. Y., Yang, L., Cowin, J. P., Iedema, M., Zhu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+and+methods+for+analyzing+liquids+under+vacuum.">Systems and methods for analyzing liquids under vacuum.</a> US Patent. , (2013).</li><li>Yu, X. Y., Liu, B., Yang, L., Zhu, Z., Marshall, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+electrochemical+device+and+process+for+chemical+imaging+and+electrochemical+analysis+at+the+electrode-liquid+interface+in+situ.">Microfluidic electrochemical device and process for chemical imaging and electrochemical analysis at the electrode-liquid interface in situ.</a> US Patent. , (2014).</li><li>Yang, L., Yu, X. Y., Zhu, Z. H., Thevuthasan, T., Cowin, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+a+hybrid+microfluidic+platform+compatible+for+in+situ+imaging+by+vacuum-based+techniques.">Making a hybrid microfluidic platform compatible for in situ imaging by vacuum-based techniques.</a> J Vac Sci Technol A. 29 (6), (2011).</li><li>Yang, L., Yu, X. Y., Zhu, Z. H., Iedema, M. J., Cowin, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+liquid+surfaces+under+vacuum+using+SEM+and+ToF-SIMS.">Probing liquid surfaces under vacuum using SEM and ToF-SIMS.</a> Lab Chip. 11 (15), 2481-2484 (2011).</li><li>Ding, Y., 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=In+Situ+Molecular+Imaging+of+the+Biofilm+and+Its+Matrix.">In Situ Molecular Imaging of the Biofilm and Its Matrix.</a> Anal Chem. , (2016).</li><li>Yang, J., Ghobadian, S., Montazami, R., Hashemi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the Asme 11th Fuel Cell Science, Engineering, and Technology Conference, 2013. , (2013).</li><li>Yu, F., Wang, C. X., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Graphene-Modified+Electrodes+in+Microbial+Fuel+Cells.">Applications of Graphene-Modified Electrodes in Microbial Fuel Cells.</a> Materials. 9 (10), (2016).</li><li>Yu, J., Zhou, Y., Hua, X., Zhu, Z., Yu, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Characterization+of+Hydrated+Proteins+in+Water+by+SALVI+and+ToF-SIMS.">In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS.</a> J Vis Exp. (108), e53708 (2016).</li><li>Hill, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Effects of Electron-Transport-System Impairment on Hydrogen Gas Production by the Bacterium Shewanella oneidensis MR-1. , (2007).</li><li>McCormick, A. J., 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=Biophotovoltaics:+oxygenic+photosynthetic+organisms+in+the+world+of+bioelectrochemical+systems.">Biophotovoltaics: oxygenic photosynthetic organisms in the world of bioelectrochemical systems.</a> Energy Environ Sci. 8 (4), 1092-1109 (2015).</li><li>Liu, B., 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=In+situ+chemical+probing+of+the+electrode-electrolyte+interface+by+ToF-SIMS.">In situ chemical probing of the electrode-electrolyte interface by ToF-SIMS.</a> Lab Chip. 14, 855-859 (2014).</li><li>Keune, K., Hoogland, F., Boon, J. J., Peggie, D., Higgitt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+"added+value"+of+SIMS:+A+mass+spectrometric+and+spectroscopic+study+of+an+unusual+Naples+yellow+oil+paint+reconstruction.">Evaluation of the "added value" of SIMS: A mass spectrometric and spectroscopic study of an unusual Naples yellow oil paint reconstruction.</a> Int J mass Spectrom. 284 (1-3), 22-34 (2009).</li><li>Lee, 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> Mass Spectrometry Handbook. , 988 (2012).</li><li>Petrovic, M., Barcelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+anionic+and+nonionic+surfactants,+their+degradation+products,+and+endocrine-disrupting+compounds+in+sewage+sludge+by+liquid+chromatography/mass+spectrometry.">Determination of anionic and nonionic surfactants, their degradation products, and endocrine-disrupting compounds in sewage sludge by liquid chromatography/mass spectrometry.</a> Anal Chem. 72 (19), 4560-4567 (2000).</li><li>Vickerman, 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=Molecular+Imaging+and+Depth+Profiling+by+Mass+Spectrometry--Sims,+MALDI+or+DESI.">Molecular Imaging and Depth Profiling by Mass Spectrometry--Sims, MALDI or DESI.</a> Analyst. 136 (11), (2011).</li><li>Weng, L. T., Bertrand, P., Stonemasui, J. H., Stone, W. E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tof+Sims+Study+of+the+Desorption+of+Emulsifiers+from+Polystyrene+Latexes.">Tof Sims Study of the Desorption of Emulsifiers from Polystyrene Latexes.</a> Surf Interface Anal. 21 (6-7), 387-394 (1994).</li><li>Pe&#241;uelas-Urquides, K., 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=Measuring+of+Mycobacterium+tuberculosis+crowth.+A+correlation+of+the+optical+measurements+with+colony+forming+units.">Measuring of Mycobacterium tuberculosis crowth. A correlation of the optical measurements with colony forming units.</a> Braz J Microbiol. 44 (1), 287-289 (2013).</li><li>Dohnalkova, A. C., 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=Imaging+hydrated+microbial+extracellular+polymers:+comparative+analysis+by+electron+microscopy.">Imaging hydrated microbial extracellular polymers: comparative analysis by electron microscopy.</a> Appl Environ Microbiol. 77 (4), 1254-1262 (2011).</li><li>Yang, L., 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=In+situ+SEM+and+ToF-SIMS+analysis+of+IgG+conjugated+gold+nanoparticles+at+aqueous+surfaces.">In situ SEM and ToF-SIMS analysis of IgG conjugated gold nanoparticles at aqueous surfaces.</a> Surf Interface Anal. 46, 224-228 (2013).</li><li>Yu, J. C., 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=Capturing+the+transient+species+at+the+electrode-electrolyte+interface+by+in+situ+dynamic+molecular+imaging.">Capturing the transient species at the electrode-electrolyte interface by in situ dynamic molecular imaging.</a> Chem Commun. 52 (73), 10952-10955 (2016).</li></ol>

저자는 공개 없다.

로그 단계에서 접종 후 수 일 및 온도를 그것은 건강 하 고 이미징, 충분히 두꺼운 3.1 단계에서 설명한 대로 전에 biofilm 성장 해야 테스트 중요 하다. 이 절차는 특히 S. oneidensis MR1 biofilm; 실 온에서 배양 커버 그러나 다른 방 온도 성장의 속도 좌우할 수 있다. 따라서, 그것은를 사용 하 여 광학 이미징는 biofilm ToF 심즈 분석을 진행 하기 전에 준비 인지 이해 중요 합니다. 마찬가지로, 박테리아의 다?...

박테리아 biofilms는 점에서 셀 연결 하 고 많은 가능한 환경에서 살아남을 수 있다 불리 한 물리적 및 기계적 자극, 다양 한 생존을 위해 박테리아에 대 한 방어로 시간이 지남에 진화 표면 관련 커뮤니티. 1 , 2 Biofilms는 광대 하 게 조사 하 고 의학, 공학, 농업, 그리고 산업 연구 및 개발 등 많은 분야에 응용 프로그램을. 1 , 2 정확 하 필수적 이다 이러한 복잡 한 미생물 지역 사회, 그들의 자체 생산된 세포 외 고분자 물질 (EPS)와 그들의 현지 물-클러스터 환경 등의 화학 매핑을 이해 하 고 상세한 그들의 생물 학적 활동 묘사 2
Biofilms는 존재 하 고 높은 수 화 상태에서 성장. 이 진공에서 휘발성 액체를 공부에 어려움으로 인해 비행 시간 2 차 이온 질량 분석 (ToF-심즈) 같은 진공 기반 표면 분석 기법을 사용 하 여 큰 도전을 선물 한다. 그 결과, 진공 기반 표면 분석 기법만 말린된 상태에서 biofilm 샘플을 공부 하 고 거의 독점적으로 제한 되었습니다. 그러나, 그것의 진정한 생물 microenvironment의 정확한 조사 억제 말린된 상태에서 biofilm을 공부. 그것은 종종 EPS 무결성 및 biofilm 형태, 건조 biofilm 제자리에 액체 연구 질량 스펙트럼 결과 비교한 후 입증 되었습니다 어떤 과감 한 변경 발생 합니다. 3 , 4 이 문서에서는 분석에는 액체 진공 인터페이스 (SALVI),5,6 미세 반응 기에 대 한 우리의 시스템의 사용을 채용 하 여 자연 수 화 상태에서 biofilms 공부에 대 한 솔루션 제공을 따라서 여전히 진공 내에서 액체 매트릭스의 구조적 무결성을 유지 하면서 보조 이온 조사 빔에 대 한 직접 액세스를 제공 하는 아닌입니다 (PMDS)의 만든에서 그것의 얇은 실리콘 나이트 라 이드 (죄) 막에서 액체를 포함 약 실입니다. 7 , 8
S. oneidensis 씨-1 녹색 형광 단백질 (GFP)를 표현 하기 위해 돌연변이이 biofilm 절차 그림 변화 다양성 및 기반 환경 및 응용 미생물학에 있는 일반적인 사용에 대 한 모델 생물으로 선정 되었다 금속 감소와 세포 외 전자 전송에 대 한 그것의 독특한 기능에 무 겁 게. 9 , 10 , 11 또한, GFP의 존재를 허용 쉽게 연속 biofilm-두께 형광 현미경 검사 법을 통해 모니터링, fluorescein isothiocyanate (FITC) 필터를 사용 하 여. 우리의 이전 연구 죄 창 최대 1 백 마이크로미터의 두께에 biofilm 성장을 위한 형광 이미징에 operando를 사용 하 여 첨부 파일을 선호 하는이 박테리아의 증거가 나타났습니다. 4 , 12 이 종이 형광 현미경 검사 법을 통해 biofilm의 존재의 확인을 토론할 것 이다, 하는 동안는 SALVI는 슈퍼 해상도 같은 다른 광학 이미징 방법에 호환 형광 이미징 (즉, 구조화 된 조명 현미경 (SIM)9) 그리고 confocal 레이저 스캐닝 현미경 검사 법 (CLSM)4이미징). 광학 이미징 biofilm 두께 측정을 제공 하 고 표시, 두께 신 창에 첨부 파일을 확인 하는 biofilm의 모양의 3D 이미지를 얻을 수 있습니다. 9 동안 GFP 심즈 분석에 사용 되었다, oneidensis S. GFP 없이 성장 곡선에 대 한 광학 밀도의이 유일한 필요한 측정으로 사용 되었다 어떤 형광 이미징 요구 하지 않았다. 일반적으로, 차이 GFP 태그 및 태그 종 성장 곡선에 중요 하지 않습니다. 또한,이 프로토콜 절차를 설명 하는 모델 생물으로 S. oneidensis 씨 1 GFP를 사용,이 절차는를 위한 SALVI 내 재배에 필요한 수 있습니다 어떤 세균성 긴장. 비록 주어진 필요한 세균성 긴장의 지식, 시간, 온도, 산소 환경 등 일부 성장 조건 사용 될 박테리아의 변형에 맞게 수정 해야 합니다. 성장 매체에 대 한이 절차는 경작에 대 한 포도 당, 포도 당 없이 tryptic 간장 agar (TSA) 없이 "나노 와이어" 중간, tryptic 간장 국물 (TSB)를 사용합니다. 성장에 대 한 고 막의 확장 모니터링에 대 한 "나노 와이어" 매체의 구성 특별히 공식화 되었습니다. 그리고 S. oneidensis 작은 전선과 중간 구성의 모양을 나타나는 periplasm 되었습니다. 이전 연구에서 설립. 13 , 14
현장에서 우리의 이전 프로토콜 액체 ToF-심즈 SALVI 단백질 immobilization와 죄, ToF 심즈 분석 및 데이터 감소에 상세한 프로토콜을 첨부 파일에 대 한 제공 하는 혜택을 설명 했습니다. 12 데이터 감소 단계를 되풀이, 보다는 오히려이 종이 될 것입니다 대신 설정 하 고 biofilm 존재와 사전 두께 감지 하는 이미징 단계 뿐 아니라 우리의 SALVI 아닌 내 biofilms 육성의 독특한 접근 방식에 초점을 ToF 심즈 분석 하. Biofilms 이전만 건조 챔버 내에 진공 기반 표면 분석 기법의 샘플을 제한 되어, 하는 동안 라이브 biofilms의 상세한 EPS 및 biofilm 화학 매핑 지금 얻을 수 있습니다 현장에서 이 새로운 기능 때문에.

<table><tbody><tr><td>ToF-SIMS</td><td>IONTOF</td><td>TOF.SIMS 5</td><td>Resolution:&gt;10,000 m/&amp;Delta;m for mass resolution;&gt;4,000 m/&amp;Delta;m for high spatial resolution</td></tr><tr><td>System for Analysis at the Liquid Vacuum Interface (SALVI)</td><td>Pacific Northwest National Laboratory</td><td>N/A</td><td>SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.</td></tr><tr><td>-80&amp;deg;C Freezer</td><td>New Brunswick Scientific</td><td>N/A</td><td>U410 Premium Energy Efficient Ultra-Low Temperature Freezer</td></tr><tr><td>4&amp;deg;C Refrigerator</td><td>BioCold Scientific</td><td>N/A</td><td>COLDBOX1</td></tr><tr><td>Orbital Shaker</td><td>New Brunswick Scientific</td><td>N/A</td><td>Innova 4900 Multi-Tier Environmental Shaker, set at 30 degrees Celsius for serum bottle and flask culturing, set at 150rpm.</td></tr><tr><td>Syringe Pump</td><td>Cole-Parmer</td><td>EW-74905-02</td><td>Cole-Parmer Syringe Pump, Infusion Only, Touchscreen Control 74905-02, used for injecting liquid into the tubing system and SALVI at a constant flowrate.</td></tr><tr><td>Incubator</td><td>Barnstead International</td><td>LT1465X3</td><td>Lab-Line incubator, set at 30 degrees Celsius for plate culturing.</td></tr><tr><td>Autoclave</td><td>Getinge</td><td>533LS</td><td>Used to sterilize PEEK fittings, tubing systems, serum vials, and medium. Model 533LS Vacuum Steam Sterilizer</td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>4001-000</td><td>GENESYS 20 spectrophotometer for OD600 readings of cuvettes for growth curves.</td></tr><tr><td>Biological Safety Cabinet</td><td>Thermo Fisher Scientific</td><td>1385</td><td>1300 Series AZ Biological Safety Cabinet</td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>N/A</td><td>Nikon OPTIPHOT-2 fluorescence microscope with camera and super high pressure mercury lamp power supply.</td></tr><tr><td>pH Meter</td><td>Mettler Toledo</td><td>51302803</td><td>Used to test the pH of the &amp;ldquo;nanowires&amp;rdquo; medium after finished and before autoclaving.</td></tr><tr><td>PEEK Union</td><td>Valco</td><td>ZU1TPK</td><td>For connecting the inlet and outlet of SALVI, the syringe to the tubing system, and the inlet of the SALVI to the drip chamber of the tubing system.</td></tr><tr><td>5 Axes Sample Stage</td><td>IONTOF</td><td>N/A</td><td>Stage is self-made for mounting SALVI in ToF-SIMS.</td></tr><tr><td>Barnstead Nanopure Water Purification System</td><td>Thermo Fisher Scientific</td><td>D11921</td><td>ROpure LP Reverse Osmosis filtration module (D2716)</td></tr><tr><td>Pipette</td><td>Thermo Fisher Scientific</td><td>21-377-821</td><td>Range: 100 to 1,000 &amp;micro;L.</td></tr><tr><td>Pipette Tip</td><td>Neptune</td><td>2112.96.BS</td><td>1,000 &amp;micro;L pipette tips</td></tr><tr><td>Razor Blade Handle</td><td>Stanley</td><td>N/A</td><td>Stanley Bostitch Razor Blade Scraper with 5 Single-Edge Blades, used for cutting PTFE tubing</td></tr><tr><td>Syringe</td><td>BD</td><td>309659</td><td>1 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>309657</td><td>3 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>309646</td><td>5 mL; Used for making the drip chamber</td></tr><tr><td>Syringe</td><td>BD</td><td>309604</td><td>10 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>302830</td><td>20 mL</td></tr><tr><td>Disposable Pipette</td><td>Thermo Fisher Scientific</td><td>13-678-11</td><td>25 mL Fisherbrand&amp;trade; Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe, for filling serum bottles.</td></tr><tr><td>Electric Pipette Filler</td><td>Pipet-aid</td><td>P-57260</td><td>Vacuum pressure electric serological pipette filler</td></tr><tr><td>Serum Bottle</td><td>Sigma</td><td>33109-U</td><td>Holds approximately 69 mL of liquid for culture growth, optimum for use of 20mL culture per bottle.</td></tr><tr><td>Anaerobic Culture Tube</td><td>VWR</td><td>89167-178</td><td>Anaerobic Tubes, 18 x 150 mm, Supplied with 20 mm Blue Butyl Rubber Stopper and Aluminum Seal.</td></tr><tr><td>Rubber Stopper</td><td>Sigma</td><td>27235-U</td><td>Silicone stopper, used for sealing serum bottles and for creating the tubing system/drip chamber.</td></tr><tr><td>Aluminum Crimp Seal (without septum)</td><td>Sigma</td><td>27227-U</td><td>Aluminum seal for top of serum bottle for use with serum bottle crimper.</td></tr><tr><td>Serum Bottle Aluminum Seal Crimper</td><td>Wheaton</td><td>224307</td><td>30 mm crimper with standard seal.</td></tr><tr><td>PTFE Tubing</td><td>Supelco</td><td>58697-U</td><td>1.58 mm OD x 0.5 mm ID 50 ft. PTFE Teflon tubing, used for creating the tubing system.</td></tr><tr><td>Disposable Cuvettes</td><td>GMBH</td><td>759085D</td><td>1.5 Ml for use with spectrophotometer.</td></tr><tr><td>Needle</td><td>BD</td><td>303015</td><td>22G; used for serum bottle injection.</td></tr><tr><td>Needle</td><td>BD</td><td>305120</td><td>23G; used for punching-through rubber stopper to create drip tubing system.</td></tr><tr><td>Shewanella oneidensis MR-1 with GFP</td><td>N/A</td><td>N/A</td><td>Matthysse AG, Stretton S, Dandie C, McClure NC, &amp;amp; Goodman AE (1996) Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145(1):87-94.&amp;nbsp;</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;S25310A</td><td>95% Denatured</td></tr><tr><td>TSA</td><td>BD</td><td>212305</td><td>Tryptic soy agar for culturing the model organism (S. oneidensis) used in this protocol</td></tr><tr><td>PIPES Buffer</td><td>Sigma</td><td>P-1851</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S-5881</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Ammonium Chloride</td><td>Sigma</td><td>A-5666</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P-4504</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma</td><td>S-9638</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher Scientific</td><td>S271-3</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium, and used to make mineral solution used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium lactate</td><td>Sigma</td><td>L-1375</td><td>60%(w/w) syrup @ 98% pure, d=1.3 g/mL, 7M, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S-5761</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nitrilotriacetic Acid Trisodium Salt</td><td>Sigma</td><td>N-0253</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Iron (III) Chloride</td><td>Sigma</td><td>451649</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma</td><td>208094</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Manganese (II) Sulfate Monohydrate</td><td>Sigma</td><td>M-7634</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Iron(II) Sulfate Heptahydrate</td><td>Sigma</td><td>215422</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma</td><td>223506</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Cobalt(II) Chloride</td><td>Sigma</td><td>60818</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Zinc Chloride</td><td>Sigma</td><td>229997</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Copper(II) Sulfate Pentahydrate</td><td>Sigma</td><td>C-8027</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Aluminum Potassium Sulfate Dodecahydrate</td><td>Sigma</td><td>237086</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Boric Acid</td><td>Sigma</td><td>B-6768</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Molybdate Dihydrate</td><td>Sigma</td><td>331058</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nickel(II) Chloride</td><td>Sigma</td><td>339350</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Tungstate Dihydrate</td><td>Sigma</td><td>14304</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>D-Biotin</td><td>Sigma</td><td>47868</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Folic Acid</td><td>Sigma</td><td>F-7876</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Pyridoxine Hydrochloride</td><td>Sigma</td><td>P-9755</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Riboflavin (B2)</td><td>Sigma</td><td>47861</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Thiamine Hydrochloride</td><td>Sigma</td><td>T-4625</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nicotinic Acid</td><td>Sigma</td><td>N4126</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>D-Pantothenic Acid Hemicalcium Salt</td><td>Sigma</td><td>21210</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Vitamin B12</td><td>Sigma</td><td>V-2876</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>4-Aminobenzoic Acid</td><td>Sigma</td><td>A-9878</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Thioctic Acid</td><td>Sigma</td><td>T-1395</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr></tbody></table>

in situ characterization of shewanella oneidensis mr1 biofilms by salvi and tof-sims

박테리아 biofilms는 그는 광대 하 게 그들의 자체 생산된 세포 외 고분자 물질 (EPS) 및 환경 미생물학에서 그들의 역할을 이해 하는 표면에 관련 된 지역 사회. 이 연구는 분석 액체 진공 인터페이스 (SALVI)에 대 한 시스템에 biofilm 첨부 파일을 육성하 고 생활 biofilm의 화학 매핑 제자리에서 비행 시간 2 차 이온 질량 분석 (ToF-심즈)에 의해 달성 하는 방법을 설명 합니다. 이 외부와 우리의 전문 설치 SALVI 채널 내의 박테리아의 경작을 통해 뿐만 아니라 biofilm 존재 및 ToF 심즈 분석 전에 두께 감지 하는 광학 이미징 기술을 통해 이루어집니다. 조각 같은 biofilm에서 크게 다른 탈수의 지역화 된 물 클러스터 환경으로 EPS에 강조 우리의 결과, 자연 수 화 상태에서 Shewanella biofilm의 특성 피크를 보여 상태입니다. 이러한 결과 진공 기반 화학 이미징 장비 biofilm 이미징 제자리에 있도록 SALVI의 획기적인 기능을 보여 줍니다.

이러한 대표적인 결과 ToF 심즈를 통해 연결 된 biofilm의 화학 프로필 수 식별 및 해석 하는 방법을 보여을 제공 합니다. ToF 심즈 데이터 수집, 절차 섹션에서 짧게 강조에서 질량 스펙트럼을 플로팅 후 피크 식별 각 각각 m/z 값에는 id를 지정 하려면 실시 한다. 이것은 박테리아와 공부, 등 다양 한 물 클러스터, 지방산, 단백질 조각 박테리아 내에서 있을 것으로 예상 되는 특?...

Watch this Scientific Journal Video about In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS at JoVE.com

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

이 문서는 제자리에서 비행 시간 2 차 이온 질량 분석 매핑의 화학 수 화 상태에서 미세 반응 기, 액체 진공 인터페이스에 분석을 위한 시스템으로 사용에 대 한 biofilm 성장을 위한 메서드를 제공 합니다. 녹색 형광 단백질으로 Shewanella oneidensis 미스터 1 모델로 사용 되었다.

제자리에서 Shewanella oneidensis MR1 Biofilms SALVI 및 ToF 심즈의 특성

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

in-situ-characterization-shewanella-oneidensis-mr1-biofilms-salvi-tof

Research

JoVE Journal

Chemistry

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

8.9K 조회수.  Pacific Northwest National Laboratory. 이 문서는 제자리에서 비행 시간 2 차 이온 질량 분석 매핑의 화학 수 화 상태에서 미세 반응 기, 액체 진공 인터페이스에 분석을 위한 시스템으로 사용에 대 한 biofilm 성장을 위한 메서드를 제공 합니다. 녹색 형광 단백질으로 Shewanella oneidensis 미스터 1 모델로 사용 되었다.

비디오: In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

소변 샘플에서 셀 무료 DNA 무결성 분석

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

연구

암 연구학

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

대량 선택 이온 소프트 랜딩에 의해 준비 잘 정의 된 표면의 제자리 SIMS 및 IR 분광법

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

화학

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

혈 청 및 혈장 복사 번호 검출 실시간 PCR를 사용 하 여

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

칩 기반 디지털 PCR에 의해 신선한 냉동 위 암 조직에서 CDH1 드문 사본 이체의 탐지

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

붕소 도핑 다이아몬드 전극 품질 및 응용 프로그램에 대한 평가<em&gt; 현장에서</em&gt; 물 전기 분해에 의해 지역의 pH 수정

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

고성능 액체 크로마토 그래피 결합 흐름 섬광에 의한 방사성 표지 및 Phosphoinositides의 세포 수준의 정량화

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

SALVI 및으로 ToF-SIMS에 의해 물에 수화 된 단백질의 제자리의 특성에

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

단백질 지 방화 Immunoblotting 또는 단백질의 분석에 대 한 애기에서 엽록체 하위 구획의 준비

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

생화학

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

제자리에서 Shewanella oneidensis MR1 Biofilms SALVI 및 ToF 심즈의 특성

요약

더 많은 비디오 탐색

이 비디오의 챕터