이 작품은 캐나다 보건 연구소 연구 (CIHR), 국립 과학 및 공학 연구위원회 (NSERC), 그리고 CIHR 교육 교부금 장학금 (LF)를 지원했습니다. 우리는 AAC를위한 교수 제라드 D. 라이트 (맥매스터 대학, 캐나다) (6) - II의 표현이 플라스미드 감사합니다.

1. 재고 솔루션을 준비 <ol><li> 관심 macromolecule 정화. (다른보고로이 경우에는 aminoglycoside N - 6&#39; - acetyltransferase - II는 (AAC6&#39; - II), 격리됩니다. 13) </li><li> 투석 버퍼의 4리터를 준비합니다. (이 경우에는 우리는 25 MM 4 사용 - (2 - 히드록시 에틸) - 1 - piperazineethanesulfonic 산 (HEPES, MW 238.3 g / 몰), 2 MM ethylenediaminetetraacetic 초산 (EDTA (에틸렌 다이아 민 테트라 초산), MW 292.2)를 포함, 산도 7.5에서.) </li><li> AAC (6 &#39;) - II 샘플 (400 μm의에서 5 ML)을 사용 투석 버퍼 (3 X 1.3 L)에 dialyzed해야 단백질 Dialyze. 최종 투석 솔루션은 기계를 씻어하고 샘플을 희석하기 위해 보관됩니다. </li><li> 0.45 μm의의 셀룰로오스 필터를 통해 최종 투석 솔루션을 필터, 4 ° C.에 저장된 &#39;실행 버퍼&#39;로 불리는 것입니다 </li><li> 실행 버퍼 철저히 씻어서되었습니다 0.2 μm의 주사기 필터를 통해 필터 단백질 솔루션입니다. 표준 검정 (브래드 포드, 로리, 등) 또는 UV 흡광도를 사용하여 단백질의 최종 농도를 측정합니다. 장기 안정성을 극대화하기 위해 단백질을 저장합니다. (AAC (6의 경우) - II 동결 융해 후 활동을 유지하지 않습니다 &#39;) - II,이 4 저장 ° C. AAC (6 의미&#39;)합니다. </li><li> 버퍼를 실행에 4.0 MG를 용해하여 아세틸 보조 효소의 25 MM 주식 솔루션 (AcCoA, 리간드, MW 809.57)의 200μL를 준비합니다. -78 ° C에서 프리즈 사용할 때까지. </li><li> 모든 단백질과 리간드 샘플 농도 무작위 표본 - 투 - 샘플 변동을 최소화하기 위해, 동일한 주식 솔루션에서 발생한해야합니다. 전체 변수 - C 세트에 걸쳐 단백질 농도에 대해 하나의 보정 계수는 분석 조정됩니다. 10 </li></ol> 2. 준비 ITC 샘플 <ol><li> 2 ML의 최종 볼륨 64 C - 값으로 효소 솔루션을 희석. (AAC (6 &#39;) - II의 경우에는이 192 μm의에 해당합니다.) </li><li> 빨리 얼음물에 리간드 (AcCoA) 재고 솔루션을 녹여. </li><li> 0.5 AcCoA 솔루션의 ML보다 10 배 농축 후이 경우에 단백질의 바인딩 사이트의 수를, 버퍼를 실행 420 μL에 AcCoA 주식 솔루션의 80 μL를 diluting을 4 MM. 준비 피펫 작성 튜브로 전송합니다. 신속 -78 ° C.에 AcCoA 주식 솔루션을 반환 </li><li> 온도 1 5 분 데가스 단백질과 진공에 따라 솔루션 ° C 원하는 실행 온도 이하 (19 ° C). </li></ol> 3. 주사기 14 설정 <ol><li> 미리 장착된 주사기 클램프는 홀더와 같은 높이가 될 때까지 주사기 홀더를 통해 사출 주사기를 삽입합니다. </li><li> 그것이 단단히 주사기 홀더의 하단에 반대 누를 때까지 주입 주사기를 통해 두 번째로 주사기 클램프 피드. 부드럽게 제공된 0.050 &quot;공 포인트 16 진수 드라이브와 클램프를 조입니다. </li><li> 피펫 홀더에 주사기 홀더 플레이스. </li><li> 부드럽게 분사 주사기로 피펫 주입기를 슬라이드. 플런저 팁이 주사기의 구멍에 직접 공급되어 있는지 확인하십시오. 일단 완전히 삽입, 피펫 주입기에 주사기 홀더의 고정 칼라를 나사. </li></ol> 4. 샘플 셀 14 로딩 <ol><li> 버퍼를 실행하는 50 ML 최소 샘플 세포를 세척하고, 오랜 needled 2.5 ML 유리 주사기를 사용하여 남아있는 액체를 제거합니다. </li><li> 천천히 청결하고 건조하게 오래 needled 2.5 ML 주사기로 단백질 샘플 솔루션의 1.8 ML 최소 그려. 모든 거품을 소개하지 않도록주의하십시오. </li><li> 조심스럽게 샘플 세포에 바늘을 삽입하고 부드럽게 세포의 땅에 닿으면. 약간 팁을 올려 (~ 1mm)과 초과 액체 샘플 세포의 맨 위에 표시됩니다 때까지 부드럽게 셀에 AAC (6 &#39;) - II 솔루션을 주입. </li><li> 오버플로의 액체 유지를 확보하면서 천천히 1cm에 대해 바늘을 올립니다. 신속하게 철회 및 샘플 세포에 갇혀있는 거품을 제거하는 솔루션의 소량 (~ 0.25 ML)를 주입. </li><li> 모든 솔루션 오버플로우를 제거합니다. 이것은 부드럽게 샘플 셀로 오버플로우의 측면을 따라 바늘을 슬라이딩에 의해 이루어진다. 주사기 팁이 난간에 타격을 줄 것으로 예상된다, 이것은 실행 솔루션을위한 욕망의 높이입니다. 이 라인 위에 앉아 모든 액체를 제거합니다. </li></ol> 5. 주사 주사기를 로딩하고 실행 14 시작 <ol><li> 입력 포트에 플라스틱 튜브 로딩 주사기를 연결합니다. </li><li> 입력 포트의 상단에 낮은 플런저 팁. </li><li> 피펫 홀더의 하단에있는 피펫 작성 튜브 AcCoA 솔루션을 놓습니다. 주사기 팁은 충전 튜브의 아래쪽을 만지지 않습니다. </li><li> 작은 금액은 로딩 주사기의 튜브를 입력하기 전까지 천천히 주사기에 솔루션을 그립니다. </li><li> 닫기 채우기 포트 버튼 플런저 팁을 클릭을 낮추는 방법으로 채우기 포트를 닫습니다. 모든 기포를 제거하기 위해 정리 -&gt; 리필 버튼을 3 번 클릭하여 정화 및 리필은 로딩하는 동안 갇혀있다가 수. </li><li> 피펫 홀더에서 작성 튜브를 제거하고 부드럽게 주사기 팁 닦으십시오. </li><li> 샘플 셀로 피펫 조립 및 부드럽게 낮은 주사기를 가져가라. 주사기 쉽게 구부 수 서서히 진행, 훌륭한 치료가 필요하는 것입니다. 주사기가 완전히 옷깃을 잠금의 기반 아래로 눌러 삽입되었는지 확인하십시오. </li><li> 원하는 실행 온도를 (이 경우 20 ° C에서) 설정하고 (이 경우 20μcal/sec)를 예상되는 최대 사출 열 흐름보다 약간 큰 참조 전원을 선택합니다. </li><li> 원하는 분사 볼륨 및 지연 프로그램. (이 경우에는, 28 주사이 고용되었습니다. 첫 번째 주사는 60의 지연 2 μL의 볼륨을했다. 모든 후속 주사는 330의 지연 10 μL의 볼륨을했다.) </li></ol> 6. 후속 실행 <ol><li> 모든 후속 실행에 2,4,8,16의 요소에 의해 AAC (6 &#39;) - II 및 AcCoA의 농도를 감소하면서, 단계 2-5를 반복하고, 32. </li></ol> 7. 데이터 분석 <ol><li> 전세계적으로 바인딩 매개 변수의 하나의 설정 등온선을 모두 맞는 맞는 프로 시저를 사용하여로 이전 10 설명했다. </li></ol> 8. 대표 결과 대표 데이터는 그림 1에 표시됩니다. 등온선의 모양은 농도에 따라 달라질 것입니다. 선명 전환이 높은에 대한 예상되는 C - 값 (즉, 높은 단백질과 리간드의 농도) (그림 2). AAC (6 &#39;) - II의 경우, 두 사이트 순차 모델 조절 stoichiometries와 동일, 독립 사이트의 두 세트를 설명하는 하나보다 더 잘 맞는를 제공합니다. <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="그림 1" /> 그림 1. AAC (6 &#39;) - II (192 μm의)에 AcCoA의 적정 (3.86 ㎜)에 의해 만들어진 등온선. A) 원시 ITC 추적. B) 2 사이트 순서에 맞게 (와 바인딩 매개 변수 (사각형) 결정에 사용되는 통합 값 -). <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="그림 2" /> AcCoA에 대한 그림 2. ITC의 등온선은 다양한 농도에서 AAC (6 &#39;) - II에 titrated. 실험 데이터 (오픈 원가) 2 세트 - 오브 - 사이트 독립적인 모델 (점선 마젠타) 및 2 현장 연속 모델 (고체 파란색)에 맞는. 2 - 사이트 sequentional 모델은 분명히 더 나은 전반적인 계약을 제공합니다. 고용 농도) 6 μm의, 0.25 MM, B) 12 μm의, 0.25 MM, C) 24 μm의 0.5 MM, D) 48 μm의, 1.0 MM, E) 96 μm의, 1.9 MM, 그리고 F) 196 μm의,되었다 각각 AAC (6 &#39;) - II 및 AcCoA에 대한 3.86 MM.

<ol>
	<li>Cliff, M. J., Ladbury, 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=A+survey+of+the+year+2002+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2002 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 16, 383-391 (2003).</li><li>Cliff, M. J., Gutierrez, A., Ladbury, 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=A+survey+of+the+year+2003+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2003 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 17, 513-523 (2004).</li><li>Ababou, A., Ladbury, 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=Survey+of+the+year+2004:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2004: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 19, 79-89 (2006).</li><li>Ababou, A., Ladbury, 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=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 20, 4-14 (2007).</li><li>Okhrimenko, O. k. s. a. n. a., J, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2006+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2006 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 1-19 (2008).</li><li>Bjelic, S., Jelesarov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2007+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2007 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 289-312 (2008).</li><li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Current Opinion in Structural Biology. 11, 560-566 (2001).</li><li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Analytical Biochemistry. 179, 131-137 (1989).</li><li>Capaldi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X-Ray+Structure+of+Zebrafish+(Danio+rerio)+Ileal+Bile+Acid-Binding+Protein+Reveals+the+Presence+of+Binding+Sites+on+the+Surface+of+the+Protein+Molecule.">The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule.</a> Journal of Molecular Biology. 385, 99-116 (2009).</li><li>Freiburger, L. A., Auclair, K., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Protein+Binding+Mechanisms+by+Variable-c+ITC.">Elucidating Protein Binding Mechanisms by Variable-c ITC.</a> ChemBioChem. 10, 2871-2873 (2009).</li><li>Wybenga-Groot, L. E., Draker, K. -. a., Wright, G. D., Berghuis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+aminoglycoside+6'-N-acetyltransferase:+defining+the+GCN5-related+N-acetyltransferase+superfamily+fold.">Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.</a> Structure. 7, 497-507 (1999).</li><li>Draker, K., Northrop, D. B., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Mechanism+of+the+GCN5-Related+Chromosomal+Aminoglycoside+Acetyltransferase+AAC(6')-Ii+from+Enterococcus+faecium:+Evidence+of+Dimer+Subunit+Cooperativity.">Kinetic Mechanism of the GCN5-Related Chromosomal Aminoglycoside Acetyltransferase AAC(6')-Ii from Enterococcus faecium: Evidence of Dimer Subunit Cooperativity.</a> Biochemistry. 42, 6565-6574 (2003).</li><li>Wright, G. D., Ladak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+characterization+of+the+chromosomal+aminoglycoside+6'-N-acetyltransferase+from+Enterococcus+faecium.">Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.</a> Antimicrob. Agents Chemother. 41, 956-960 (1997).</li><li>MicroCal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ITC Data Analysis in Origin. , (2004).</li></ol>

관심 없음 충돌 선언하지 않습니다.

가변 C 피팅이 분석 부분은 이전에 자세하게 설명 10했습니다. 여기 우리는이 접근 방식에 적합한 가변 - C 데이터 세트를 수집 실용적인 측면을보고합니다. 그것은 모든 단백질과 리간드 샘플이 동일한 주식 솔루션에서 도출되는 중요합니다. 따라서 충분한 재고 솔루션 실험 시리즈 전체를 완료하는 데 처음에 준비하는 것이 중요합니다. 이것은 AAC의 비율 (6 &#39;) - II를 보장하고 AcCoA 모든...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetyl coenzyme A (AcCoA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2056</td>
<td>&nbsp;</td>
<tr>
<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7365-45-9</td>
<td>&nbsp;</td>
<tr>
<td>ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431788</td>
<td>&nbsp;</td>
<tr>
<td>Spectra/Por 2 Dialysis Tubing</td>
<td>&nbsp;</td>
<td>Spectrum Labs</td>
<td>132678</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Syringe Filter (0.2 &mu;m)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>281445-477</td>
<td>&nbsp;</td>
<tr>
<td>Cellulos Nitrate Membrane Filters (0.45 &mu;m)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>7184-004</td>
<td>&nbsp;</td>
<tr>
<td>VP-ITC</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>VP-ITC</td>
<td>Microcalorimeter used for measurements</td>
</tr>
<tr>
<td>ThermoVac</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>USB Thermo Vac</td>
<td>Temperature Controlled Degassing Station</td>
</tr>		</tbody>
		</table>
		</div>

collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

등온 적정의 열량 (ITC)는 일반적으로 호스트 macromolecule에 리간드의 바인딩과 관련된 열역학적 파라미터를 결정하는 데 사용됩니다. ITC는 호스트 / 리간드 상호 작용을 공부에 대한 일반적인 spectroscopic 방법을 통해 몇 가지 장점이 있습니다. 예를 들어, 공개 또는 두 구성 요소가 상호 작용할 때 흡수 열을 직접 측정 및 외인성 기자가 필요하지 않습니다. 따라서 바인딩 엔탈피 및 협회 상수 (카)가 직접 ITC 데이터에서 얻은되며, entropic 기여를 계산하는 데 사용할 수 있습니다. 또한, 등온선의 모양이 C - 가치와 관련된 기계론의 모델에 따라 달라집니다. C - 값으로 정의 C = N [P] tKa 어디로 [P] t는 단백질 농도이며, 여기서 n은 호스트 내에서 리간드 바인딩 사이트의 번호입니다. 대부분의 경우, 특정 리간드에 대해 여러 개의 바인딩 사이트가 아닌 동등하고 ITC는 개별 바인딩 사이트에 대한 열역학적 바인딩 매개 변수의 특성을 허용합니다. 이것은 그러나 올바른 바인딩 모델을 사용해야합니다. 다른 모델은 동일한 실험 데이터를 끼워 넣을 수있는 선택이 문제가 될 수 있습니다. 우리는 이전에이 문제가 C - 값 몇 가지의 실험을 수행하여 피할 수 것으로 나타났습니다. C - 값이 다른에서 얻은 여러 등온선은 별도의 모델을 동시에 맞게됩니다. 올바른 모델은 다음 전체 변수 - C의 데이터 세트에 걸쳐 적합의 장점을 기반으로 식별됩니다. 이 과정은 aminoglycoside 내성 유발 효소 aminoglycoside N - 6&#39; - acetyltransferase - II (AAC (6 &#39;) - II) 여기에 적용됩니다. 우리의 방법론은 어떤 시스템에 적용할 수 있지만,이 전략의 필요성은 더 allostery 또는 cooperativity를 보여주는 macromolecule - 리간드 시스템 시연, 그리고 다른 바인딩 모델은 동일한 데이터에 본질적으로 동일한 맞는를 제공하면됩니다. 우리의 지식, 상용 그런 시스템은 없습니다. AAC (6 &#39;) - II는 두 subunits 사이 cooperativity을 보여주는이 활성화된 사이트를 포함하는 호모 - 이합체입니다. 그러나 C - 값이 하나에서 얻은 ITC 데이터는 적어도 두 가지 모델이 두 세트 - 오브 - 사이트 독립적인 모델과 두 사이트를 연속 (협동) 모델 똑같이 잘 맞지 수 있습니다. 위의 설명과 같이 C - 가치를 변화를 통해, 그것은 AAC에 대한 올바른 바인딩 모델 (6 &#39;) - II는 두 사이트를 순차적으로 결합 모델입니다 설립되었습니다. 여기에, 우리는 ITC의 실험을 수행할 때 가변 C 분석에 적합한 데이터 세트를 얻기 위해 이동해야하는 단계를 설명합니다.

Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

ITC는 호스트에 리간드의 바인딩을 연구를위한 강력한 도구입니다. 복잡한 시스템에서 그러나, 몇 가지 모델은 동일하게 데이터를 맞을 수 있습니다. 방법은 여기에 설명된 것은 복잡한 시스템에 적합한 바인딩 모델을 명료하게하다와 해당 열역학적 파라미터를 추출하는 수단을 제공합니다.

바인딩 메커니즘을 결정하기 위해 가변 농도 등온 적정의 열량의 데이터 세트를 수집

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

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Biology

12.7K 조회수.  McGill University. ITC는 호스트에 리간드의 바인딩을 연구를위한 강력한 도구입니다. 복잡한 시스템에서 그러나, 몇 가지 모델은 동일하게 데이터를 맞을 수 있습니다. 방법은 여기에 설명된 것은 복잡한 시스템에 적합한 바인딩 모델을 명료하게하다와 해당 열역학적 파라미터를 추출하는 수단을 제공합니다.

비디오: Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Immunoperoxidase 분석을 사용하여 인간 Coronaviruses의 적정

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

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생물학

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

외과 듀엣으로 Nociceptive 및 염증성 중재자를 수집 및 측정

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

단백 분해 효소의 활동을 확인하는 프로 테아제 형광 검출 키트의 사용

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR - 클립 - RNA 단백질의 바인딩 Transcriptome 전체 바인딩 사이트를 식별하는 방법

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Macromolecule - 리간드 친화력을 측정하기위한 등온 적정의 열량

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

덕트 세포를 수집 배양 차 쥐 내부 수질

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

글리코겐의 생화학 적정<em&gt; 체외</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

형광 기반 프라이머 확장 기술은 전사 시작 포인트 및 RNases의 쪼개짐 사이트를 확인하는<em&gt; 생체</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.