이 연구는 ISF, NSF, 그리고 ERC에 의해 지원되었다. 우리는 랜디 밀포드, 마이클 코렌, 덕 Shafer, 그리고 제레미 데니슨의 온도 단계에 대한 기술적 인 도움을 인정하고 싶습니다. 소프트웨어 개발 지원은 또는 첸, 디 슈, Rajesh Sannareddy, 그리고 Sumit Bhattachary에 의해 제공되었다. 우리는 MP IBP의 단백질과 도움이 토론을 보려면 공동 교수 피터 L. 데이비스 박사 로리 A. 그라함 감사드립니다. 우리는 또한 감사 실험실 구성원 박사 마야 바 - Dolev, Yangzhong 진 박사 Yeliz Celik, 닥터 나탈리아 Pertaya, Ortal Mizrahy, 그들의 사용자 의견 Shlomit 사람.주세요

0. 예비 절차 <ol><li> 유리는 솔루션 주입을위한 모세관. 모세 풀러을 (Narishige, 도쿄, 일본)를 사용, 유리 모세관 마이크로 튜브 (브랜드 GMBH, Wertheim, 독일)에서 좋은 시작과 함께 날카로운 피펫을 준비합니다. 개방의 크기는 깨끗한 물에서 고급 버블 링을 얻기 위해 모세관을 통해 공기를 전달하여 확인해야합니다. 모세가 닫혀 있다면, 하나는 가장자리를 위반하여 열 수 있습니다. 이것은 튜브 벽을 포함하는 물에 대해 누르거나 부드럽게를 긁적에 의해 수행 될 수있다. 개방 거의 차단되어 있지만 충분히 하위 mm 거품의 형성을 허용하도록 열어되어있는 모세관 등을 준비합니다. </li><li> 구리 디스크 청소. 더블 증류수로 씻어 후, 0.1 % 마이크로-90 비누 (콜 - Parmer, 버논 힐스, 일리노이, 미국)의 10 분의 구리 디스크를 Sonicate. 이소프로판올 (기술) 솔루션으로 디스크를 소개하고 10 분에 다시 sonicate. 지느러미동맹국, 필터링 공기를 사용하여 디스크를 건조. 이 청소 단계는 실험 사이의 IBP 오염을 방지하는 것이 중요합니다. </li><li> 더블 레이어 coverglass 조립. coverglass 조립은 커버 유리 표면에 수분을 응축하지 않고 샘플 관찰을 할 수 있도록 준비되었습니다. 이것은 다음 뜨거운 아교 총 접착 두개가 coverslips 사이 Drierite (WA 해먼드 Drierite, 크세 니아, 오하이오, 미국) 입자 (직경 2mm)를 배치에 의해 달성되었다. 이 구성 예제는 낮은 온도로 냉각하고 관찰 창에 건조 공기를 날려 할 필요가 제거되었을 때 시야를 가리지 수 결로하지 못했습니다. </li></ol> 1. 냉각 단계 설정 <ol><li> 4mm 내경 Tygon 튜브 (생 Gobain, 파리, 프랑스)로 냉각 단계의 물 흐름 입구와 출구를 연결하고, 워터 펌프로 물 흐름 입구 관을 연결합니다. </li><li> 냉각 단계 t의 입구에 4mm 내경 Tygon 튜브를 연결건조한 공기를 제공 O. 공기가 인라인 Drierite 열을 사용하여 건조되었습니다. </li><li> 공기와 물 펌프를 운영하고 있습니다. 냉각 요소가 히트 싱크없이 실행하지 말아야합니다. </li><li> 온도 컨트롤러, 카메라, LabVIEW 루틴을 사용합니다. </li></ol> 2. 샘플 준비 <ol><li> 디스크를 통해 교련 500 μm 구멍을 갖는 7mm 직경의 구리 디스크의 뒷면에 집중 기름 B (Cargille 실험실, 삼나무 로브, 뉴저지, 미국)의 3-4 μl 소적를 놓습니다. </li><li> 아래로 향한 집중 오일 측과 냉각 무대에서 구리 디스크를 배치합니다. </li><li> 2 ML 유리 주사기 (Poulten-그라프, Wertheim, 독일)의 다른 쪽 끝에서 연결 0.7 mm 내경 Tygon 튜브에 모세관 튜브를 (무딘 가장자리)에 연결합니다. </li><li> 이전 모세관 튜브를 사용하기 시작합니다 (예비 절차를 참조) 적절한 크기인지 확인하기 위해 모세관의 작은 구멍을 확인합니다. </li><li> 천천히 gla를 삽입SS은 준비된 IBP 단백질 샘플 튜브 (산도 8시 20 밀리미터에서 2.4 μM MP IBP-GFP CaCl 2, 25 MM 트리스 - HCL, 준비 대한 자세한 내용은 참조 10 참조)으로 모세관과 모세관 유리가 0.1이 포함되어 때까지 유리 주사기를 당겨 단백질 솔루션 μl. </li><li> LabVIEW 소프트웨어를 통해 비디오 녹화를 시작합니다. </li><li> 냉각 무대에서 구리 디스크에있는 구멍 중 하나에 모세관 유리 (단백질 솔루션을 포함)의 날카로운 가장자리를 삽입합니다. </li><li> (올림푸스, 도쿄, 일본, 10X 목적) 현미경으로 관찰하는 동안주의 깊게 유리 모세관 팁과 집중 기름 층을 관통하고, 단백질의 소량 (~ 10 NL)를 제공하기 위해 유리 주사기를 (아주 섬세) 키를 눌러 200 μm의 비말을 만드는 솔루션입니다. </li><li> 더블 레이어 coverglass 조립체 (예비 절차 참조) 냉각 단계에서 구멍을 다룹니다. </li></ol> 3. TH 활동 측정 <ol><li> 사전ss가 냉각 버튼을 -40로 온도를 설정 ° C. </li><li> 처음에는 솔루션 비말가 명확하게됩니다. 낮은 온도에서 일반적으로 범위 -30 ° C -35에 대한 해결책이 고정되었음을 나타내는 ° C, 비말의 색상이 변경됩니다. 대량 얼음이 녹기 시작할 때까지 샘플 얼려 후 즉시 천천히 온도를 높일 수 있습니다. 온도의 점진적인 증가는 샘플의 전체 녹는 될 수있는 온도의 오버 슈팅을 방지 할 필요가 있습니다. </li><li> 50x 목표로 전환하여 온도를 조절하여 얼음을 녹기 시작합니다. 이 조정은 대화 형이며, 마지막 단계는 일반적으로 0.002 ° C.의 작은 온도 단계를 사용하여 수행됩니다 하나의 크리스탈이 남아 때까지 녹기를 계속합니다. 크리스탈의 마지막 크기는 10 μm 주변에 있어야합니다. 용해가 중단 한되는 최고 온도는 융점로 결정되며 나중에 비디오 분석 단계에서 정확하게 결정됩니다. </li> &lt;리&gt; 크리스탈의 녹는 점 아래 섭씨 학위를 몇 hundredths로 온도를 설정하고 10 분 지연 온도 램프를 시작합니다. 원하는대로 ramping 속도를 조정합니다. 이 기간 동안 크리스탈은 IBPs에 노출 될 것입니다. </li><li> 10 분 노출 시간이 완료되면, 온도는 LabVIEW 루틴의 제어하에 자동으로 줄어 듭니다. </li><li> 온도 감소로 결정 모양을 관찰합니다. 어떤 시점에서, 얼음 크리스탈의 갑작스러운 폭발이 관찰 될 수 있습니다. 이 문제가 발생되는 온도는 크리스탈 버스트 온도로 기록됩니다. </li><li> 정확한 녹는 점과 버스트 온도를 결정하기 위해 비디오 분석을 사용합니다. 첫째, 비디오 분석을 사용하여, 정확한 녹는 점을 찾으십시오. 용해가 중단 한되는 최고 온도가 녹는 점으로 결정됩니다 연상케합니다. 스프레드 시트 프로그램에서이 녹는 점을 문서화. 그런 다음 정확한 크리스탈 버스트 온도를 결정하고,이 값을 문서화뿐만 아니라. 녹는 점과 어는점, 또는 크리스탈 버스트 온도, 사이의 차이점은 IBP 솔루션의 열 이력의 활동입니다. </li></ol> 4. 시간에 따라 달라 TH 활동의 측정 <ol><li> 얼음의 단일 크리스탈을 준비하는 제 3.1-3.3에 설명 된 프로토콜을 따르십시오. </li><li> 크리스탈 형성 한 후, 원하는대로 진입로를의 지연 시간을 설정하고, 램프를 켜십시오. </li><li> 온도가 자동으로 한 번 램프 지연 시간이 경과 (사업자의 요구에 따라) 고정 비율로 감소합니다. </li><li> 크리스탈 버스트가 발생되는 온도를 문서화. 노출 시간 (크리스탈 형성과 크리스탈 버스트 사이의 시간) 계산합니다. </li><li> 다양한 지연 시간에 대한 실험을 반복하고 TH 활동의 시간 의존도를 평가하는 노출 시간의 함수로 TH 활동을 하겠나. </li></ol>

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L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+experiments+reveal+that+antifreeze+proteins+bound+to+ice+crystals+suffice+to+prevent+their+growth.">Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 1309-1314 (2013).</li><li>Middleton, A. J., Marshall, C. B., Faucher, F., Bar-Dolev, M., Braslavsky, I., Campbell, R. L., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+protein+from+freeze-tolerant+grass+has+a+beta-roll+fold+with+an+irregularly+structured+ice-binding+site.">Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.</a> J. Mol. Biol. 416, 713-724 (2012).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 작품은 특별한 온도 제어 TH 활동의 정확한 측정을 가능하게하는 컴퓨터 제어 nanoliter의 osmometer의 작동을 보여줍니다. 어떤 온도에 민감한 시스템에서 원치 않는 온도 기울기는 피해야합니다. 여기에 제시된 장치에서 온도 기울기를 방지하려면 테스트 솔루션 물방울이 구리 디스크 냉각 단계 (단계 2.7)의 구멍의 중심에 위치해야합니다. 또한, 단일 크리스탈은 비말의 중심에보다는 가장자리 근처에 있어야합니다 (대부분의 경우에이 저절로 발생합니다.) 설명 시간 의존성은 냉각 속도가 TH의 판독에 영향을 미칠 수 있음을 나타냅니다. 따라서, 우리는 결정을 냉각하기 전에 솔루션에 노출 된 기간 동안 시간에 대한 보고서뿐만 아니라, 냉각 속도 등의하시기 바랍니다. 우리는 일반적으로 0.01에서 온도를 내려 ramping하기 전에 10 분을 기다렸 ° C 각 4 초 단계를 반복합니다. LabVIEW 제어 공동oling 단계는 마이크로 유체 장치는 열 조작 할 수있는 역 현미경으로 사용하기 위해 적응되었다. 이 시스템은 eGFP 9, 10, 16로 태그가 얼음 결정과 IBPs를 포함하는 솔루션 교환 실험의 성능을 용이하게한다. LabVIEW 제어 시스템은 지정된 적응 전기 회로를 통해 3040 온도 컨트롤러를 연결하여 클리프톤 단계에 적용 할 수 있습니다. 이러한 시스템은 데이비스 연구소 17에 운영하고 있습니다. LabVIEW 소프트웨어와 클리프 튼 단계에 대한 지정 적응 전기 회로 설계는 요청시 이용 가능합니다. 결론적으로, 우리는 중요한 제어 및 조작 온도 및 실시간 분석을위한 LabVIEW 루틴을 통해 비디오 인터페이스를 조정 온도 증가와 감소의 속도 (0.002 ° C 감도 포함), 촉진 nanoliter의 osmometer을 설명합니다. 이 시스템은 importan 아르 재현 속도 제어 실험을 수행 할 수 있습니다얼음과 IBP 상호 작용의 동역학을 조사에 대한 t. 이러한 실험 IBPs의 행동의 메커니즘을 둘러싼 여러 장기 논쟁 문제를 해결 할 수 있습니다.

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> 카탈로그 번호 / 모델 </td><td> 코멘트 </td></tr><tr><td> 집중 오일 타입 B </td><td> Cargille 연구소 </td><td> 16,484 </td><td></td></tr><tr><td> Drierite </td><td> WA 해먼드 Drierite </td><td> 043,063 2,270g </td><td></td></tr><tr><td> 마이크로 90 청소 솔루션 </td><td> 콜 - Parmer </td><td> <a href="http://www.coleparmer.com/Product/Cole_Parmer_small_sup_reg_sup_small_Micro_90_small_sup_reg_sup_small_cleaning_solution_case_of_four_4_Liter_bottles/EW-18100-11" target="_blank">EW-18100-11</a> </td><td></td></tr><tr><td> 풀러을 모세관 </td><td> Narishige </td><td> PB-7 </td><td></td></tr><tr><td> 유리 모세관 튜브 </td><td> 브랜드 GNBH </td><td> 7493 21 </td><td> 길이 75mm, 직경 1.15 </td></tr><tr><td> 온도제어 장치 </td><td> 뉴 포트, 어빈, 캘리포니아, 미국 </td><td> 모델 3040 </td><td> 모델 3040 </td></tr><tr><td> 빛 현미경 </td><td> 하늘 </td><td> 모델 BH2 </td><td></td></tr><tr><td> 10X 목표 </td><td> 하늘 </td><td></td><td> S 계획 10, 0.3, 160/0.17 </td></tr><tr><td> 50x 목적 </td><td> 니콘 </td><td></td><td> CF 계획, 50X/0.55 에피 ELWD </td></tr><tr><td> CCD 카메라 </td><td> Provideo </td><td> CVC-140 </td><td></td></tr><tr><td> Tygon 튜브 </td><td> 생 Gobain, 파리, 프랑스 </td><td></td><td> Tygon 제제 S-50-HL 관 </td></tr><tr><td> 유리 주사기 (2 ML) </td><td> Poulten - 그라프, Wertheim, 독일 </td><td> 7 10,227 </td><td></td></tr><tr><td> GPIB-PCI 카드 </td><td> 내쇼날 인스트루먼트, 오스틴, 텍사스, 미국 </td><td> 778032-01 </td><td></td></tr><tr><td> 비디오 프레임 GRAbber IMAQ-PCI-1407 </td><td> 내쇼날 인스트루먼트, 오스틴, 텍사스, 미국 </td><td> 322156B-01 </td><td></td></tr><tr><td> LabVIEW 시스템 디자인 소프트웨어 </td><td> 내쇼날 인스트루먼트, 오스틴, 텍사스, 미국 </td><td> 버전 8 </td><td></td></tr><tr><td> DiVx 저자 소프트웨어 </td><td> DiVx LLC, 샌디에고, CA, USA </td><td></td><td></td></tr></tbody></table>

labview-operated novel nanoliter osmometer for ice binding protein investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

TH 시간 의존성 측정 LabVIEW 운영하는 nanoliter의 osmometer 정확한 TH 활동 측정의 성능을 용이하게한다. 일정한 온도 감소 속도는 TH 시간 의존성의 측정을 사용할 수 있습니다. nanoliter의 osmometer으로 활성화 정확한 온도 제어는 이러한 실험에 대한 중요한했습니다. 솔루션에 IBPs에 얼음 결정의 노출 시간은 크리스탈 (크리스탈 버스트) 주변 얼음의 갑작스런 성장 때까지 크리스탈의 형성에서 기간 (녹는 과정의 끝)로 정의됩니다. 우리는 IBPs에 얼음 결정의 노출 시간이 결정적인 TH 활동에 영향을 것으로 나타났습니다. IBP 노출 (몇 초)의 짧은 기간은 MP IBP-GFP 솔루션 (2.4 μM) (그림 5)에서 낮은 TH 활동을 생산. 그 4 분 IBP 노출의 고원에 도달 할 때까지 TH 활동은 IBP 노출 시간 증가했다. 높은 IBP 농도에서, 판AU는 짧은 시간에 도달했습니다. <img alt="그림 1" src="/files/ftp_upload/4189/4189fig1.jpg" /> 그림 1. 도식 다이어그램 도시 IBPs은 얼음으로 adsorbed. 10 일부터 허가를 채택했다. <img alt="그림 2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> 그림 2. 냉각 무대. A) 현미경에 튜브로 연결되어 있습니다. B) 위의 리드없이. C) 도식 다이어그램. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank"> </a><img alt="그림 3" src="/files/ftp_upload/4189/4189fig3.jpg" /> 그림 3. LabVIEW 인터페이스의 스크린 샷. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">망할 CIA</a>큰 그림을 보려면 여기를 ick. <img alt="그림 4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> 그림 4. 온도 안정성 그래프. 온도 컨트롤러는 온도 0.01 ° C 매 15 초를 내려하도록 설정되었습니다. <img alt="그림 5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> 그림 5. IBPs에 얼음 크리스탈 노출 시간의 함수로 MP IBP의 TH 활동. 때마다 포인트 3-6 실험의 평균입니다.

Watch this Scientific Journal Video about LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations at JoVE.com

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

또한 부동액 단백질로 알려진 아이스 바인딩 단백질 (IBPs)는, 얼음의 성장을 억제하고 조직의 cryopreservation에서 사용할 수있는 유망 첨가제입니다. IBPs을 조사하는 데 사용되는 주요 도구는 nanoliter의 osmometer입니다. 우리는 광학 현미경에 장착 홈 디자인 냉각 단계를 개발하고 맞춤형 LabVIEW 루틴을 사용하여 제어. nanoliter의 osmometer는 매우 민감한 방식으로 샘플 온도를 조작 여기에 설명했다.

아이스 바인딩 단백질 수사 LabVIEW 운영하는 소설 Nanoliter의 Osmometer

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

labview-operated-novel-nanoliter-osmometer-for-ice-binding-protein

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JoVE Journal

Chemistry

20.3K 조회수.  The Hebrew University of Jerusalem. 또한 부동액 단백질로 알려진 아이스 바인딩 단백질 (IBPs)는, 얼음의 성장을 억제하고 조직의 cryopreservation에서 사용할 수있는 유망 첨가제입니다. IBPs을 조사하는 데 사용되는 주요 도구는 nanoliter의 osmometer입니다. 우리는 광학 현미경에 장착 홈 디자인 냉각 단계를 개발하고 맞춤형 LabVIEW 루틴을 사용하여 제어. nanoliter의 osmometer는 매우 민감한 방식으로 샘플 ?...

비디오: LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

단일 분자의 단백질 연구에 대한 표면 패시베이션

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

연구

화학

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

공유 결합은 자기 조립 단분자막의 접근 방식을 사용하여 표면에 BMP-2의 결합

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

형광 기반 얼음 비행기 선호도에 의해 부동액 단백질의 얼음 결합 평면 결정

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

단백질 - 단백질 상호 작용 및 알로 스테 릭 리간드 효과의 속도론을 측정하는 바이오 층 간섭

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

말라리아 단백질 바이오 마커 히스티딘 풍부한 단백질-II의 검출을위한 이리듐 (III) 발광 프로브

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

오일 유제에서 물 : 소수성 안료와 수용성 엽록소 결합 단백질을 조립하기위한 새로운 시스템

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

ELISA 기반 바인딩과 경쟁 방법은 신속 리간드 - 수용체 상호 작용을 확인하는 방법

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE - 지우기 기본 젤 - 용출 액체 분수 내포 전기 영동

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

EOB-DTPA와의 조지아 (III) 단지에 대한 조사 그것<sup&gt; (68)</sup&gt; 조지아 방사성 표지 된 아날로그

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

Photoresponsive 액정의 구조 역학을 관찰을 위한 새로운 기술

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...