우리는 그녀의 데이터를 사용하기 위해 베이징 우 감사합니다. 이 작품은 JEK에 NSF 경력 보너스에 의해 지원되었다

1. 막 단백질 폴딩에 대한 ~ 50 나노미터 직경 소형 Unilamellar Vesicles (포드 SUV)의 준비 <ol><li> 1,2 - dimyristoyl - SN - glycero - 3 - phosphocholine (DMPC) 클로로포름에 lipids의 솔루션은 스토리지를위한 유리병 수량 당 20 밀리그램에서 구입한 깨끗한 유리 튜브에 aliquotted입니다. 질소 가스 층은 지질 산화를 방지하기 위해 각 유리병에 추가하고, 유리병은 뚜껑과 parafilm과 함께 봉인하고 있습니다. 튜브는 사용 전까지 -20 ° C의 냉동고에 저장됩니다. </li><li> 클로로포름 20 MG의 지질의 나누어지는를 포함 하나의 약병은 각 실험에 사용됩니다. 유리병의 내용은없이 용매 남아 때까지 1 시간 동안 질소 가스의 흐름을 사용하여 건조합니다. </li><li> 말린 lipids 20 MM의 칼륨 인산 버퍼 1 ML에 resuspended 아르 ~ 30 초 동안 물 목욕 sonicator를 사용하여 (산도 = 7.3). 정지 지방질의이 솔루션은 흐림 나타납니다, 그리고 원뿔 바닥과 플라스틱 15 ML 튜브로 전송됩니다. 인산염 버퍼의 또 다른 한 ML의 나누어지는 원래 지질을 포함 빈 유리관에 추가하고, sonication이 반복되며 솔루션은 다음 같은 15 ML 튜브에 추가됩니다. 튜브의 마지막 볼륨이 재고 소포 솔루션 5 MG / ML의 지질 농도의 결과, 4 ML 때까지이 과정은 4 회 총 반복됩니다. </li><li> 지질 소포 솔루션의 4 ML 볼륨에 배치됩니다 따뜻한 (~ 30 ° C) 물 목욕, 및 50 % 듀티 사이클에서 1 시간 ultrasonicator의 microtip를 사용하여 sonicated. 물 목욕의 목적은 두 가지입니다. 첫째, 그것은 때문에 sonication 프로세스의 너무 뜨거운되는에서 지질 솔루션을 방지할 수 있습니다. 둘째, 따뜻한 목욕은 수성 지질 솔루션의 온도가 sonication 동안 이중층 위상 전이 온도 이상 유지 보장, DMPC 들어,이 전이 온도는 ~ 23 ° C. </li><li> sonicated 솔루션은 sonicator 팁에서 파편을 제거하고 필터링 솔루션은 37 하룻밤 equilibrated입니다 필터 0.22 μm의 주사기 ° C. 통해 전달된다 </li></ol> 2. 초기 형광 펼쳐 곡선에 대한 샘플 준비 <ol><li> 20 MM의 인산 버퍼 (산도 7.3) 10 M의 요소의 주식 솔루션은 이루어집니다. 이 요소의 많은 수량이 솔루션에서 중요한 볼륨을 차지하고 있기 때문에 물이 고체 (아니라 그 반대)에 추가하는 것이 중요합니다. 이 요소 솔루션은 깨끗하고, 빈 병 solutes를 추가하고 의도한 최종 볼륨에 물을 추가하여 만들 수 있습니다. 이 솔루션은 물 목욕이나 용질을 용해하는 감동과 함께 뜨거운 접시에 온난 화를 요구할 수 있습니다. 요소는 흡습이므로, 우레아 주식 솔루션의 실제 농도는 굴절률을 사용하여 결정한다 1. </li><li> 20 MM 인산 (산도 7.3)의 주식 버퍼 솔루션은 이루어집니다. </li><li> 의 주식 솔루션은 단백질이 준비가되어 펼쳐진 거죠. 이 주식 단백질 솔루션 8 M의 요소, 20 MM 인산 버퍼에서 ~ 200 μm의 단백질의 농도가 있어야합니다. </li><li> 형광 연구에 대한 샘플은 다음과 같은 방법으로 준비하고 있습니다. 재고 단백질의 적절한 볼륨 (섹션 2.3), 주식 지질 솔루션 (5 MG / ML 지질, 제 1 항), 주식 10 M의 요소 솔루션 (섹션 2.1), 주식 인산 버퍼 (섹션 2.2)은 ~ 4를 포함하는 샘플을 만들어 결합 μm의 단백질, 1 MG / ML 지질, 1 M의 단위 0-8 M의 요소. 각 샘플의 총 부피는 200 μL입니다. 예를 들어 스프레드 시트 (표 1) 목록의 볼륨이 200 μm의 주식 단백질 솔루션을 사용하는 데 필요한 것이 포함되어 있습니다. </li><li> 빈 샘플 섹션 2.4에서 설명한대로 만들어진, 그러나, 단백질이 추가되지 않습니다. 다른 구성 요소의 농도 섹션 2.4들에게 동일하므로 단백질 대신에, 8 M의 요소 솔루션 4 μL를 추가할 수 있습니다. 이러한 공백은 산란과 단백질의 형광 스펙트럼에 나타나는 다른 배경 신호를 빼야하는 데 사용됩니다. </li><li> 섹션 2.4와 2.5의 샘플이 완성 접는와 평형을 보장하기 위해 형광 스펙트럼 측정하기 전에 최소한 2 시간 동안 37 ° C에서 부화 사용할 수 있습니다. </li></ol> 3. 형광 스펙트럼 측정 각각의 샘플 및 빈의 트립토판의 형광은 정상 상태 fluorometer를 사용하여 측정됩니다. 형광 스펙트럼은 티로신 잔류물의 여기를 피하기 위해 290 nm의 파장의 여기로 기록된하고, 305-500 nm의에서 스캔해야합니다. 일반 입구와 출구 밴드는 3 NM입니다. 파장 단위 및 통합 시간은 신호 대 잡음 비율에 대해 최적화할 수 있습니다. 파장 단위 및 통합 시간에 대한 일반적인 값은 1 NM / 단계 각각 0.5 초 / 단계입니다. 막은 온도가 이중층 위상 전이 온도 이상의 경우에만 합성 lipids에 일반적으로 배 단백질. 따라서, DMPC의 경우, 시료의 온도는 30 일정 열립니다 ° C.를 160 μL 용량 microvolume 융합 실리카 쿠베트는이 전직에 활용periments. 4. 펼쳐 막 단백질의 깁스 자유 에너지의 초기 전개 곡선 및 견적 생성 <ol><li> XY의 데이터 세트의 형태로 형광 스펙트럼은 같은 이고르 프로, Matlab, 유래, 또는 Excel과 같은 소프트웨어에로드됩니다. </li><li> 요소 및 지질 소포 배경에서 신호가 다음 절차를 사용하여 빼서해야합니다 지질 vesicles 및 요소의 특정 농도의 존재에있는 단백질의 A = 원시 형광 스펙트럼 스펙트럼. 스펙트럼 B 지질 vesicles와 같은 요소 농도를 포함 해당 빈 샘플의 형광 스펙트럼 = 원료 것과 스펙트럼에서, 어떤 단백질이 비어 샘플에 없습니다. 수정된 스펙트럼 = 스펙트럼 - C * 스펙트럼 B. C가 1.0으로 일반적으로 동일 스칼라입니다. 어떤 경우에는, C 때문에 원인이 산란 문제 이하 1.0보다 큰 이상 또는 ~ 305-320 nm의 영역에 가까운 빼기 이하. </li><li> 최대 형광, λ 최대의 파장은 각 요소 농도에 대한 수정 스펙트럼에서 tabulated 있습니다. 완벽에 대한 일반적인 가치는 접힌 단백질의 그 ~ 330 nm의 상태에서 막 단백질 350 NM입니다 펼쳐진 거죠. Tabulated 파장가 펼쳐졌 인구의 분율에 λ 최대의 가치를 상호 연관시킬 1.0 범위 0.0에 λ 최대 값 (~ 350 nm의로 ~ 330)의 범위를 변환하여 펼쳐 분수로 변환됩니다. 예를 들면, 330 nm의 최대의 λ 값이 0.0에 해당하는 350 nm의 1.0에 해당하고, 340 nm의이 분수의 관점에서 0.5에 해당 펼쳐진 거죠. 펼쳐 단백질의이 분수는 다음 요소 농도 역모를 꾸몄다입니다. 위에서 언급한 바와 같이, 요소의 농도는 굴절률을 측정하여 실험적으로 결정되어야합니다. </li><li> 우리는 단백질이 두 가지 상태 중 하나에 존재 할 수 있다고 가정합니다 : 접으면 또는 펼쳐진 거죠. 분수의 줄거리는 F, 펼쳐, 대 요소의 농도, C는 방정식에 맞게 수 : 2 <img src="/files/ftp_upload/2669/2669eq2.jpg" alt="방정식 2" /> 계수 m은 denaturant 농도와 관련하여 자유 에너지의 변화의 속도에 해당하고, C M은 접힌 인구가 펼쳐 인구와 동일되는 미드의 요소 농도에 해당합니다. 이 값은 R은 (0.001987 kcal / 몰 • K) 가스 법률 상수입니다. 데이터 분석 소프트웨어를 사용하여 최소 제곱 피팅에서 얻은 피팅 변수이며, T는 (303 K) 온도입니다. </li><li> 맞지에서 얻은 계수는 요소의 부재에서 펼쳐의 깁스 자유 에너지 (kcal / 몰) 결정하기 위해 활용됩니다 <img src="/files/ftp_upload/2669/2669eq3.jpg" alt="방정식 3"/> . </li></ol> 5. 펼쳐의 깁스 자유 에너지의보다 정확한 측정 곡선을 펼쳐 최적화. <ol><li> 위에서 설명한 펼쳐 커브가 λ 최대의 가장 큰 변화를 일으키는 요소의 농도의 범위를 나타낸다. 이 범위는 일반적으로 소형이며, 전개의 대부분이 작은 범위 내에서 발생합니다. 펼쳐의 자유 에너지를 정확하게 결정하기 위해 추가 샘플은 최적화된 최소 제곱 피팅에 필요한 많은 데이터 포인트를 포함하는 음모를 얻기 위해이 지역에서 분석하고 있습니다. 보다 정확하게 펼쳐 곡선이 중요한 지역의 모양을 나타내기 위해 0.2 M의 증가 4 M의 요소 - 단백질 2 및 4 M의 요소 사이에 펼쳐집니다 예를 들어, 샘플 2가 포함되어있다는 사실을 만들 수 있습니다. 그것은 각 요소의 농도에서 빈 스펙트럼을 측정하기 위해 필요하지 않습니다, 그것은 모든 ~ 0.5 M 단계 빈 스펙트럼을 측정하기 충분하다. </li><li> 이 음모를 얻은 펼쳐의 자유 에너지는 원래의 전개 곡선에서 얻은 것과 약간 다를 수 있지만,보다 정확한 가치를 반영 수 있습니다. </li></ol> 6. 대표 결과 : <table border="1"><tbody><tr><td> 견본 </td><td> 마지막 [요소], M </td><td> 재고 단백질 (μL) </td><td> 재고 지질 (μL) </td><td> 재고 10 M 우레아 (μL) </td><td> 주식 버퍼 (μL) </td></tr><tr><td> 1 </td><td> ~ 0.16 </td><td> 4 </td><td> 40 </td><td> 0 </td><td> 156 </td></tr><tr><td> 2 </td><td> 1 </td><td> 4 </td><td> 40 </td><td> 20 </td><td> 136 </td></tr><tr><td> 3 </td><td> 2 </td><td> 4 </td><td> 40 </td><td> 40 </td><td> 116 </td></tr><tr><td> 4 </td><td> 3 </td><td> 4 </td><td> 40 </td><td> 60 </td><td> 96 </td></tr><tr><td> 5 </td><td> 4 </td><td> 4 </td><td> 40 </td><td> 80 </td><td> 76 </td></tr><tr><td> 6 </td><td> 5 </td><td> 4 </td><td> 40 </td><td> 100 </td><td> 56 </td></tr><tr><td> 7 </td><td> 6 </td><td> 4 </td><td> 40 </td><td> 120 </td><td> 36 </td></tr><tr><td> 8 </td><td> 7 </td><td> 4 </td><td> 40 </td><td> 140 </td><td> 16 </td></tr></tbody></table> 표 1. 형광 샘플을하기 위해 필요한 재고 솔루션 볼륨 <img src="/files/ftp_upload/2669/2669fig1.jpg" alt="그림 1" /> 그림 1. 단일 트립토판 성분을 포함하고 ~ 5 μm의 대표 막 단백질의 트립토판 형광 스펙트럼. 단백질의 (A) 생 형광 스펙트럼 (스펙트럼 4.2에서) 빈의 (B) 생 형광 스펙트럼은 (4.2에서 스펙트럼 B) (C) 4.2에서 스펙트럼을 교정. <img src="/files/ftp_upload/2669/2669fig2.jpg" alt="그림 2" /> 그림 2. 다양한 요소의 농도에 대한 그림 1에서 막 단백질의 트립토판 형광 스펙트럼을 교정. <img src="/files/ftp_upload/2669/2669fig3.jpg" alt="그림 3" /> 그림 3. 4.4 방정식에 맞게으로 그림 2에있는 데이터에서 얻은 곡선을 펼쳐. 깁스 자유 에너지는 섹션 4.5에 따라 계산됩니다.

<ol>
	<li>Shirley, B. A., Shirley, B. 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> Protein Folding and Stability. , 177-190 (1995).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+and+Analysis+of+Urea+and+Guanidine+Hydrochloride+Denaturation+Curves.">Determination and Analysis of Urea and Guanidine Hydrochloride Denaturation Curves.</a> Methods Enzymol. 131, 266-279 (1986).</li><li>Lau, F. W., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+Assessing+the+Stability+of+a+Membrane+Protein.">A Method for Assessing the Stability of a Membrane Protein.</a> Biochemistry. 36, 5884-5892 (1997).</li><li>Burgess, N. K., Dao, T. P., Stanley, A. M., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Barrel+Proteins+that+Reside+in+the+Escherichia+coli+Outer+Membrane+in+Vivo+Demonstrate+Varied+Folding+Behavior+in+Vitro.">&#946;-Barrel Proteins that Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro.</a> J. Biol. Chem. 283, 26748-26758 (2008).</li><li>Booth, P. J., Curnow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+scene+investigation:+membrane+proteins.">Folding scene investigation: membrane proteins.</a> Curr. Opin. Struct. Biol. 19, 8-13 (2009).</li><li>Hong, H., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+coupling+of+integral+membrane+protein+stability+to+lipid+bilayer+forces.">Elastic coupling of integral membrane protein stability to lipid bilayer forces.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 4065-4070 (2004).</li><li>Sanchez, K. M., Gable, J. E., Schlamadinger, D. E., Kim, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Tryptophan+Microenvironment,+Soluble+Domain,+and+Vesicle+Size+on+the+Thermodynamics+of+Membrane+Protein+Folding:+Lessons+from+the+Transmembrane+Protein+OmpA.">Effects of Tryptophan Microenvironment, Soluble Domain, and Vesicle Size on the Thermodynamics of Membrane Protein Folding: Lessons from the Transmembrane Protein OmpA.</a> Biochemistry. 47, 12844-12852 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

현재 프로토콜은 트립토판 잔류물을 포함하는 막 - 관련 단백질과 펩티드의 곡선을 펼쳐의 생성을 설명합니다. 여기, 그것은 트립토판의 형광이 단백질이 접힌 및 합성 지질 vesicles에 삽입하거나, 용액에 펼쳐진 여부를 반영하는 것으로 간주됩니다. 이러한 두 상태 폴딩 및 denaturant 농도와 자유 에너지의 선형 의존성과 같은 추가 가정은, 현재의 보고서에서 만들어집니다,. 다른 방정식에서 이러?...

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<td>DMPC</td>
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<td>Avanti Polar Lipids</td>
<td>850345C</td>
<td>&nbsp;</td>
<tr>
<td>Urea</td>
<td>&nbsp;</td>
<td>MP Biochemicals</td>
<td>04821527</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Phosphate Dibasic</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P288</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Phosphate Monobasic</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P285</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

thermodynamics of membrane protein folding measured by fluorescence spectroscopy

막 단백질 접힘은 기본적인 건강 관련 모두 의미있는 새로운 주제입니다. 세포의 underlies 단백질이 유비 쿼터스 가족의 접는 종합 학습에 대한 필요성의 막 단백질의 풍부. 또한, misfolded 단백질과 관련된 질병의 특징 우리의 방위 능력에 대한 발전은 단백질 폴딩의 분야에서 상당한 실험 및 이론적인 노력을 동기 있습니다. 이 중요한 분야에서 급속한 진전은 불행하게도 막 단백질과 접는 메커니즘의 복잡과 관련된 고유의 도전에 의해 방해합니다. 여기, 우리는 E. 대표 일체형 멤브레인 단백질에 대한, denaturant, Δ G ° H 2 O의 부재에서 펼쳐의 깁스 자유 에너지의 열역학적 특성을 측정하기위한 실험 절차를 설명합니다 대장균. 이 프로토콜은 denaturant 농도의 함수로 접힌 및 펼쳐진 상태의 평형 인구를 결정하기 위해 형광 분광법의 응용 프로그램에 초점을 맞추고 있습니다. 합성 지질 vesicles뿐 아니라 데이터 분석 절차의 주요 단계의 준비를 위해 실험적인 고려 사항이 강조 표시됩니다. 이 기술은 다양한이며 온도와 산도,뿐만 아니라 lipids과 micelles의 다양한 환​​경을 포함하여 접는 denaturant의 종류와 추구있을 수 있습니다. 현재 프로토콜은 아래에 설명되어 기준 집합을 충족하는 막이나 가용성 단백질에 일반 수있는 하나입니다.

Watch this Scientific Journal Video about Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy at JoVE.com

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

트립토판 형광으로 막 단백질 폴딩의 깁스 자유 에너지를 얻기 위해이 동영상 기사 세부 실험 절차.

막 단백질 폴딩의 열역학은 형광 분광학에 의해 측정

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

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18.2K 조회수.  University of California San Diego - UCSD. 트립토판 형광으로 막 단백질 폴딩의 깁스 자유 에너지를 얻기 위해이 동영상 기사 세부 실험 절차.

비디오: Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

microfluidic 비말 플랫폼 형광 검출 방법

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

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생체공학

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

단백질 접기 유학을위한 Microfluidic 믹서

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

막 척추 : 막 단백질의 단백질 - 단백질 상호 작용을 식별 할 수있는 생화학 적 도구<em&gt; 생체</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

수정 된 두 개의 광자 현미경에서 3D 궤도 추적 : 세포 내 소포의 추적에 응용

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

브릴 루앙 분광학에 대한 세포 외 기질 단백질 섬유의 제조

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

정식이 아닌 아미노산의 선택적 압력 관에 의해 '황금' 형광 및 질량 분석 및 형광 단백질 분석 엔지니어링

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

나노 제형에도 Limulus Amoebocyte Lysate (LAL) 분석 실험을 사용 하 여

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

자율 적인 소포 성장을 위한 펩티드 멤브레인 전구체의 베시큘로 합성

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

생체멤브레인 포스 프로브 분광법에 의한 분자 스프링 상수 분석

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How &quot;Molecular Surgery&quot; of the Backbone Affects Folding and Stability

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

형광 단백질에서 프롤린 유사체에 의한 프롤린의 잔기 특이적 교환 : 백본의 "분자 수술"이 폴딩 및 안정성에 미치는 영향

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...