Name: ensemble force spectroscopy by shear forces
Uploaded: 2022-07-26T13:00:00
Description: Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

이 연구 작업은 국립 과학 재단 [CBET-1904921] 및 국립 보건원 [NIH R01CA236350]의 지원을 받았습니다.

참고: 이 프로토콜에 사용된 모든 완충액과 화학 시약은 표 재료에 나열되어 있습니다.
1. 전단력 현미경의 제조
참고: 전단력 현미경은 반응 장치(균질화기)와 검출 장치(형광 현미경)의 두 부분으로 구성됩니다. 접안 렌즈의 배율은 10 배이고 대물 렌즈 (공기)의 배율은 4 배입니다.
<ol><li>균질화기와 현미경을 장착 테이블에 조?...

<ol>
	<li>Neuman, K. C., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+force+spectroscopy:+Optical+tweezers,+magnetic+tweezers+and+atomic+force+microscopy.">Single-molecule force spectroscopy: Optical tweezers, magnetic tweezers and atomic force microscopy.</a> Nature Methods. 5 (6), 491-505 (2008).</li><li>Woodside, M. T., 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=Nanomechanical+measurements+of+the+sequence-dependent+folding+landscapes+of+single+nucleic+acid+hairpins.">Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (16), 6190-6195 (2006).</li><li>Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+strong+is+a+covalent+bond.">How strong is a covalent bond.</a> Science. 283 (5408), 1727-1730 (1999).</li><li>Strick, T. R., Allemand, J. F., Bensimon, D., Croquette, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+supercoiled+DNA.">Behavior of supercoiled DNA.</a> Biophysical Journal. 74 (4), 2016-2028 (1998).</li><li>Yang, D., Ward, A., Halvorsen, K., Wong, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+single-molecule+force+spectroscopy+using+a+centrifuge.">Multiplexed single-molecule force spectroscopy using a centrifuge.</a> Nature Communications. 7, 11026 (2016).</li><li>Su, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-responsive+polymer+particles+as+force+clamps+for+the+mechanical+unfolding+of+target+molecules.">Light-responsive polymer particles as force clamps for the mechanical unfolding of target molecules.</a> Nano Letters. 18 (4), 2630-2636 (2018).</li><li>Kirkness, M. W. H., Forde, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+assay+for+proteolytic+susceptibility:+Force-induced+collagen+destabilization.">Single-molecule assay for proteolytic susceptibility: Force-induced collagen destabilization.</a> Biophysical Journal. 114 (3), 570-576 (2018).</li><li>Astumian, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+and+kinetics+of+molecular+motors.">Thermodynamics and kinetics of molecular motors.</a> Biophysical Journal. 98 (11), 2401-2409 (2010).</li><li>Bekard, I. B., Asimakis, P., Bertolini, J., Dunstan, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+shear+flow+on+protein+structure+and+function.">The effects of shear flow on protein structure and function.</a> Biopolymers. 95 (11), 733-745 (2011).</li><li>Chistiakov, D. A., Orekhov, A. N., Bobryshev, Y. V. <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+shear+stress+on+endothelial+cells:+go+with+the+flow.">Effects of shear stress on endothelial cells: go with the flow.</a> Acta Physiologica. 219 (2), 382-408 (2017).</li><li>Hu, C., Jonchhe, S., Pokhrel, P., Karna, D., Mao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+unfolding+of+ensemble+biomolecular+structures+by+shear+force.">Mechanical unfolding of ensemble biomolecular structures by shear force.</a> Chemical Science. 12 (30), 10159-10164 (2021).</li><li>Sedghi Masoud, S., 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=Analysis+of+interactions+between+telomeric+i-motif+DNA+and+a+cyclic+tetraoxazole+compound.">Analysis of interactions between telomeric i-motif DNA and a cyclic tetraoxazole compound.</a> ChemBioChem. 19 (21), 2268-2272 (2018).</li><li>Abraham Punnoose, 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=Adaptive+and+specific+recognition+of+telomeric+G-quadruplexes+via+polyvalency+induced+unstacking+of+binding+units.">Adaptive and specific recognition of telomeric G-quadruplexes via polyvalency induced unstacking of binding units.</a> Journal of the American Chemical Society. 139 (22), 7476-7484 (2017).</li><li>Dhakal, S., 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=Coexistence+of+an+ILPR+i-motif+and+a+partially+folded+structure+with+comparable+mechanical+stability+revealed+at+the+single-molecule+level.">Coexistence of an ILPR i-motif and a partially folded structure with comparable mechanical stability revealed at the single-molecule level.</a> Journal of the American Chemical Society. 132 (26), 8991-8997 (2010).</li><li>Hu, C., Tahir, R., Mao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+mechanochemical+sensing.">Single-molecule mechanochemical sensing.</a> Accounts of Chemical Research. 55 (9), 1214-1225 (2022).</li></ol>

이 원고에 설명 된 프로토콜을 사용하면 전단력에 의한 생체 분자 구조의 앙상블 세트의 전개를 실시간으로 조사 할 수 있습니다. 여기에 제시된 결과는 DNA i- 모티프 구조가 전단력에 의해 펼쳐질 수 있음을 강조합니다. 리간드 결합 i-모티프의 풀림과 전단력 작동 클릭 반응은 이 앙상블 힘 분광법의 개념 증명 응용 분야였습니다.
그림 1 은 기기 설정을...

단일 분자 힘 분광법1(SMFS)에서 개별 분자 구조의 기계적 특성은 원자력 현미경, 광학 핀셋 및 자기 핀셋 2,3,4와 같은 정교한 도구에 의해 연구되었습니다. 힘 생성/검출 설정에서 분자의 동일한 방향성 요구 사항 또는 자기 핀셋 및 소형 원심분리기 힘 현미경(MCF)5,6,7,8의 작은 시야로 인해 제한되므로 SMFS를 사용하여 제한된 수의 분자만 동시에 조사할 수 있습니다. SMFS의 낮은 처리량은 많은 분자 세트의 개입을 필요로하는 분자 인식 분야에서의 광범위한 적용을 방해합니다.
전단 흐름은 거대한 분자 세트에 힘을 가하는 잠재적 솔루션을 제공합니다9. 채널 내부의 액체 흐름에서, 채널 표면에 가까울수록, 유량(10)은 느려진다. 이러한 유속 구배는 경계 표면에 평행한 전단 응력을 유발합니다. 분자가 이 전단 흐름에 배치될 때, 전단력이 장축(11)에 가해짐에 따라, 분자는 그 장축이 유동 방향과 정렬되도록 스스로 방향을 바꾼다. 이러한 방향 변경의 결과로 동일한 유형(핸들의 크기 및 길이)의 모든 분자가 동일한 전단력을 경험하면서 동일한 방향으로 정렬될 것으로 예상됩니다.
이 작업은 DNA i- 모티프에 의해 예시 된 바와 같이 거대한 분자 구조 세트에 전단력을 가하기 위해 이러한 전단 흐름을 사용하는 프로토콜을 설명합니다. 이 프로토콜에서는 균질화기 팁에서 회전자와 고정자 사이에 전단 흐름이 생성됩니다. 본 연구는 접힌 DNA i- 모티프 구조가 9724-97245 s-1의 전단 속도에 의해 펼쳐질 수 있음을 발견했다. 게다가, L2H2-4OTD 리간드와 i-모티프 사이에 36μM의 해리 상수가 발견되었다. 이 값은 겔 시프트 분석12에 의해 측정된 31 μM의 값과 일치한다. 또한, 현재 기술은 클릭 반응을 촉매하기 위해 킬레이트화된 구리(I)를 노출시킬 수 있는 i-모티프를 전개하는데 사용된다. 따라서 이 프로토콜을 사용하면 합리적인 시간(30분 미만)에 저비용 악기를 사용하여 대규모 i-motif 구조 세트를 펼칠 수 있습니다. 전단력 기술이 힘 분광법의 처리량을 크게 증가시킨다는 점을 감안할 때 이 기술을 앙상블 힘 분광법(EFS)이라고 합니다. 이 프로토콜은 이 전단력 기반 EFS의 적용을 용이하게 하기 위한 실험 지침을 제공하는 것을 목표로 합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3K MWCO Amicon</td>
			<td>Millipore Sigma</td>
			<td>ufc900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>VWR</td>
			<td>VWRC0143-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calfluor 488 azide</td>
			<td>Click Chemistry Tools</td>
			<td>1369-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CuCl</td>
			<td>Thermo&#160;</td>
			<td>ACRO270525000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispersion tip</td>
			<td>Switzerland</td>
			<td>PT-DA07/2EC-B101</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA oligos</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dye</td>
			<td>IDT</td>
			<td>/5Cy5/</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Janpan</td>
			<td>Nikon TE2000-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Switzerland</td>
			<td>PT 3100D</td>
			<td></td>
		</tr>
		<tr>
			<td>HPG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>cs-295271</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>VWRC26760.295</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>VWR</td>
			<td>VWRCE169-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quencher</td>
			<td>IDT</td>
			<td>/3IAbRQSp/</td>
			<td></td>
		</tr>
		<tr>
			<td>TBTA</td>
			<td>Tokyo Chemical Industry</td>
			<td>T2993</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>VWRCE133-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

ensemble force spectroscopy by shear forces

형광 및 기계화학적 원리에 기반한 단일 분자 기술은 생물학적 감지에서 우수한 감도를 제공합니다. 그러나 높은 처리량 기능이 없기 때문에 이러한 기술의 적용은 생물 물리학에서 제한적입니다. 앙상블 힘 분광법(EFS)은 개별 분자의 기계화학적 연구를 분자 앙상블의 연구로 변환하여 방대한 분자 구조 세트의 조사에서 높은 처리량을 입증했습니다. 이 프로토콜에서 DNA 2차 구조(i-모티프)는 최대 77796/s의 전단 속도로 균질화기 팁의 회전자와 고정자 사이의 전단 흐름에서 펼쳐졌습니다. 유속과 분자 크기가 i-모티프가 경험하는 전단력에 미치는 영향이 입증되었습니다. EFS 기술은 또한 DNA i- 모티프와 리간드 사이의 결합 친화도를 밝혀 냈습니다. 또한, 우리는 전단력(즉, 기계-클릭 화학)에 의해 작동될 수 있는 클릭 화학 반응을 시연했습니다. 이러한 결과는 분자 구조의 형태를 제어하기 위해 전단력을 사용하는 효과를 입증합니다.

그림 1은 EFS에서 앙상블 분자의 기계적 풀림 및 실시간 감지를 간략하게 설명합니다. 도 1B에서, i-모티프 DNA의 형광 강도는 pH 5.5 MES 완충액에서 9,724 s-1 내지 97,245 s-1 범위의 전단 속도에 따라 증가하는 것으로 관찰되었다. 대조군으로서, 동일한 i-모티프 DNA를 pH 7.4 MES 완충액에서 63,209 s-1의 속도로 전단했을 때 형광...

Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

Ensemble Force Spectroscopy by Shear Forces

앙상블 힘 분광법(EFS)은 생물물리학 및 생물감지 분야에서 생체 분자 구조의 앙상블 세트의 기계적 풀림 및 실시간 감지를 위한 강력한 기술입니다.

전단력에 의한 앙상블 힘 분광법

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the ...

이 프로토콜은 DNA와 리간드 사이의 분자간 결합을 연구하기 위한 고처리량 기술을 제공합니다. 이 기술은 방대한 생체 분자 구조 세트의 조사에서 높은 처리량을 입증했습니다. 개별 분자의 기계적 화학적 특성을 분자 앙상블의 기계적 특성으로 변환함으로써 그렇게 합니다.나는 그것이 매우 진보 된 실험이라고 생각하지만 비판적인 단계는 거의 없습니다. 예를 들어, 현미경, 균질화기 및 반응 튜브의 정렬. 형광의 변화를 정확하게 추적하기 위해서는 이 세 가지가 완벽하게 정렬되어야 합니다.먼저 균질화기와 현미경을 장착 테이블에 조립합니다. 고글을 착용한 후 형광 현미경을 켠 다음 균질화기를 조정하여 여기 광선이 균질화기의 분산 팁 중심을 통과하는지 확인합니다. 그런 다음 높이가 5cm, 단면적이 1.5 x 1.5 평방 센티미터인 평평한 바닥 반응 챔버를 준비하여 선택한 챔버 재료가 형광을 발하지 않도록 하여 배경을 줄입니다.그 후, 형광 현미경의 샘플 스테이지에 반응 챔버를 고정한 다음 균질화기의 분산 팁을 유지하도록 챔버를 조정하여 팁이 반응 챔버의 바닥 표면보다 약간 위에 있는지 확인합니다. 다음으로, 균질화기와 챔버의 수직 위치를 함께 조정하여 현미경의 초점이 분산 팁의 표면에 오도록 합니다. 그런 다음 분산 팁의 수평 위치를 조정하여 로터와 스테이터 사이에 감지 영역이 설정되었는지 확인합니다.실험에 사용된 형광 염료에 따라 형광 채널을 켭니다. 고속 전단 실험 전에 탈 이온수를 사용하여 낮은 전단 속도로 전단을 테스트했습니다. 먼저, 원고에 기재된 바와 같이 탈이온수에서 각각 염료 및 소광제로 표지된 인간 텔로미어 i-모티프 DNA를 탈이온수에서 준비한다.다음으로, DNA를 pH 5.5 또는 pH 7.4에서 30 밀리몰 MES 완충액에서 5 마이크로몰로 희석한다. DNA 용액에 0에서 60 마이크로 몰 범위의 리간드 L2H24OTD "를 첨가하고 용액을 10 분 동안 부드럽게 혼합하여 빛없이 i- 모티프 구조를 접습니다. 탈이온수로 채워진 반응 챔버의 배경 형광 강도를 전단 없이 형광 현미경을 사용하여 확인하고 최소화합니다.그런 다음 노출 시간이 0.5 초, CCD 감도가 1, 600, 녹화 시간이 20 분인 소프트웨어를 사용하여 CCD 카메라의 매개 변수를 설정하십시오. 긴 피펫을 사용하여 DNA 용액을 비어 있고 깨끗한 반응 챔버에 넣고 반응 챔버를 블랙 박스로 덮습니다. 그런 다음 균질기를 시작하여 CCD 카메라를 켜서 데이터를 기록하기 위해 20 분 동안 초당 9, 724에서 초당 97, 245 범위의 선택된 전단 속도로 전단을 수행합니다.실험 후, 챔버를 제거하고 탈 이온수로 세척한다. i-모티프 DNA와 탈이온수를 준비합니다. 이어서, 10 마이크로몰 i-모티프 DNA 및 150 마이크로몰 염화구리 및 300 마이크로몰 아스코르브산이 보충된 300 마이크로몰 트리스 완충액을 10분 동안 인큐베이션하여 i-모티프 구조를 접는다.다음으로 울트라 여과 장치로 용액을 14, 300 배의 원심력으로 G. 각 여과 후 300 마이크로 몰 아스코르브 산이 보충 된 30 밀리몰 트리스 완충액으로 용액을 약 500 마이크로 리터로 보충합니다. 잔류 용액을 수집 한 후 300 마이크로 몰 아스코르브 산이 보충 된 30 밀리몰 트리스와 20 마이크로 몰 CalFluor 488 Azide, 20 마이크로 몰 HPG 및 10 마이크로 몰 TBTA를 추가하여 300 마이크로 리터의 최종 부피를 만듭니다. 시약이 추가되면 용액을 암실로 옮깁니다.그런 다음 전단 실험 전에 현미경을 사용하여 탈이온수로 채워진 반응 챔버의 배경 형광 강도를 확인하고 최소화합니다. 다음으로, DNA 용액을 긴 피펫으로 빈 반응 챔버에 넣은 다음 CCD 카메라를 켠 상태에서 20 분 동안 초당 63, 209의 전단 속도로 균질 기 전단을 시작합니다. 실험 후, 챔버를 제거하고 탈 이온수로 세척한다.i-motif DNA의 형광 강도는 pH 5.5 MES 완충액에서 초당 9, 724에서 초당 97, 245 범위의 순전한 속도로 증가하는 것으로 관찰되었다. 그러나, 동일한 i-motif DNA가 MES 완충액에서 pH 7.4에서 접히지 않기 때문에 초당 63, 209의 속도로 전단되었을 때 형광 강도는 증가하지 않았다. 순전히 흐름을 거친 DNA i-모티프 분자에 대한 리간드 결합을 평가한 결과, 리간드 농도가 증가함에 따라 형광 강도의 증가가 덜 분명해진다는 것을 보여주었습니다.해리 상수는 리간드와 i-모티프 사이의 상호작용에 대해 결정되었고, 34 마이크로몰로 밝혀졌다. 구리 1 킬레이트 i- 모티프는 초당 63, 209 전단 속도로 펼쳐졌으며, 방출 된 구리 하나는 형광 클릭 반응을 유발하여 시간이 지남에 따라 형광 강도가 증가했습니다. 먼저 로터와 스테이터 사이에 감지 영역이 설정되어 있는지 확인하십시오.둘째, 형광 배경을 확인하고 최소화합니다. 마지막으로 클릭 반응을 수행하기 전에 반응을 필터링해야 합니다. 이 방법은 모든 종류의 폴리머를 접고 펼치는 기계적 강도를 탐색하는 관문을 엽니다.다른 DNA 구조, 단백질 또는 기타 폴리머 및 다른 종류의 분자와의 상호 작용 일 수 있습니다. 말했듯이 이 접근법을 사용하여 몇 가지 다른 거대분자를 연구할 수 있습니다.

ensemble-force-spectroscopy-by-shear-forces

Research

JoVE Journal

Bioengineering

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

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 ...

연구

생체공학

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

원자 힘 현미경에 의해 영상에 대한 세균 고정

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

SynVivo 합성 미세 혈관의 네트워크를 사용하여 전단 접착지도의 생성

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

높은 가위 균질화 및 시리얼 농도로 농축, 지질 기반 산소 마이크로 버블 에멀젼의 제조

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

투과성이 골격 근육 섬유에 의해 생성 된 최대 아이소 포스의 측정

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

원자 힘 현미경 이미징 및 지원 지질 이중층의 힘 분광학

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

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

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

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

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

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

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

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 ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

점성-관성 포스 제팅을 번갈아 가며 생체외 하이드로겔 마이크로캐리어의 3D 프린팅

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...