이 연구는 NIDCR의 교내 연구 프로그램에 의해 지원되었다.

1. 도식 개요 : 이 동영상은 입술 분석을 수행에 관련된 단계를 설명합니다. 항원은 재조합 Renilla의 루시페라제 (Ruc) 항원 fusions, 그리고 원유 추출물로 Cos1 세포에 표현 취득하고 정화없이 사용. 입술 분석은 microtiter 우물에서 환자 세라와 크루드 전자 Ruc - 항원 추출물을 잠복기로 시작됩니다. 항체 - 항원 혼합물은 다음 IgG 분자를 캡처하는 단백질 A / G 비즈를 포함하는 96 - 웰 플레이트 필터로 전송됩니다. 단백질을 포함하고있는 필터 플레이트 세척 후 Ruc - 항원이 coelenterazine 기판 및 조명 장치의 추가에 의해 측정된다 바운드 A / G 구슬, 항체는 luminometer로 측정됩니다. 1 부 : Renilla - 항원 융합 단백질의 생산을위한 Plasmids의 Transfection 설정 : Cos1 세포가 표준 조직 문화 프로토콜을 사용하여 DMEM - 10% FCS에서 양식입니다. Renilla 루시페라제 fusions에 대한 Plasmids는 이전에 다음 ID로 한 설명했습니다. 이러한 plasmids에 대한 DNA는 Qiagen에서 Midiprep 키트를 사용하여 준비가되어 있습니다. 수율은 약 1 -3 MG해야합니다. -20 ° C.에서 1,000 μg / ML 주식 솔루션으로 DNA 농도 및 저장을 측정 절차 : <ol><li> 하루 전에 transfection, 분할 있으니까요 - 1 개의 새 항목 100 37 판과 부화 당 약 2 X 10 6 X 20mm 요리 ° C.에 세포 </li><li> 다음날, 왜냐하면 - 1 세포가 80~95% 합류한다. 각각의 플라스미드 DNA에 대한 레이블 1.5 ML의 폴리 프로필렌의 microfuge 튜브는 transfected 수 있습니다. 4 저장되어있는 FuGENE - 6 transfection 시약, ° C는, 적당한 온도로 워밍업 수 있습니다. </li><li> 각 microfuge 튜브에 Opti - 멤 미디어의 94 μl를 추가합니다. 다음 측면 벽을 건드리지 않고도 Opti - 멤 미디어 FuGENE 6 개 중 6 μl를 추가합니다. </li><li> 상온에서 5 분 혼합물을 품어. </li><li> 1-2 Renilla 루시페라제 항원 융합에 대한 플라스미드의 μg (1mg/ml의 DNA 재고에서) 구조를 추가합니다. 혼합 후 실온에서 15 분 동안 혼합물을 품어. </li><li> Cos1 세포의 미디어에 고르게 드리핑하여 세포에 DNA - FuGENE 6 Opti - 멤 솔루션을 전송합니다. </li></ol> 2 부 : 착취 Renilla - 항원 Fusions <ol><li> 이틀 transfection 후, 왜냐하면 - 1 세포가 수확됩니다. 이것은 미디어를 제거하고 PBS 6 ML과 세포 rinsing에 의해 시작됩니다. PBS를 decanting 후, 조직 문화 요리에서 잔여 PBS 멀리 피펫. </li><li> 50 MM 트리스, 산도 7.5, 100 MM NaCl, 5 MM MgCl 2, 1 % 트리톤 X - 100, 50 % 글리세롤과 테아제 억제제 (50 ML 당 전체 miniprotease 억제제 칵테일 2 정제 구성된 차가운 용해 버퍼의 1.4 ML 추가 용해 버퍼). 휴대 scrapper로 수확 세포가 빠르게 얼음에 두 개의 1.5 ML의 microfuge 튜브 각 lysate의 절반을 전송할 수 있습니다. </li><li> 브렌스 Sonifier 150 세포가 열려 휴식하는 데 사용됩니다. 각각, 2, 2, 4 sonication 설정으로 5 초, 5 초 5 초위한 얼음과 펄스에있는 세포 lysate를 포함하는 microcentrifuge 튜브를 놓습니다. </li><li> 첫 번째 스핀 후, 부드럽게 튜브의 측벽에서 느슨하게 연결된 파편을 제거하기 위해 튜브를 반전 네 두 사분 돌지 ° C. 위해 12,500 RPM의 세포 lysate를 원심 분리기. 두 번째 스핀 후, 얼음에 새로운 microfuge 튜브에 두 튜브에서 펠렛을 방해하지 않고, 표면에 뜨는를 주의깊게 전송합니다. </li><li> lysate의 μl 당 빛의 단위 (LU)를 계산합니다. LU를 측정하기 위해, 새로운 microfuge 튜브에 PBS 8 μl와 lysate 1 μl를 희석. 직접 희석 혼합에 1X coelenterazine 기판 100 μl를 추가하고 즉시 오초 읽을 수있는 튜브 luminometer을 (20분의 20 N 터너 과학)를 사용하여 튜브의 발광을 측정합니다. </li><li> -80에서 aliquots의 시대의 긴 기간 1-2 일 또는 저장소에 대해 -20 ° C에서 Ruc - 항원 lysate를 저장 ° C. </li></ol> 3 부 : 세라 마스터 플레이트를 준비 <ol><li> 첫번째 (50 MM 트리스, 산도 7.5, 100 MM NaCl, 5 MM MgCl 2, 1 % 트리톤 X - 100) 96 깊은 잘 폴리 프로필렌의 microtiter 플레이트의 각 잘하는 버퍼의 450 μl를 추가하여 세라 마스터 플레이트 만들기 . 이 단계에서, 염료, 페놀 레드는 또한 미래 세라 샘플 추가 및 입술 분석의 다른 단계를 모니터링을위한 추적 역할을 (최종 농도가 버퍼에서 0.2 μg / ML)을 버퍼에 포함될 수 있습니다. </li><li> 다음 버퍼 A.의 450 μl를 포함하는 다른 우물 각 샘플에서 세라의 50 μl를 추가할 이것이 일반적으로 세라가 마스터 판의 마지막 두 우물에 추가되지 않습니다 버퍼 A.에서 세라의 1시 10분 희석 참고하기 때문에 이것은 버퍼 공백에 사용됩니다. </li><li> 마스터 플레이트를 사용하기 전에, 그것은 광범위한 회전 플랫폼 (1~2시간) 흥분 상태입니다. 마스터 플레이트에 혈청 역입니다증발을 방지하기 위해 올바르게 저장 ° C의 경우 4 최소한 1 개월 (또는 이상)의 경. </li><li> 아래에 설명한 바와 같이,이 마스터 플레이트 반복 여러 항원에 대항하는 세라의 프로파일 링을 위해 제거하는 희석 세라 10 μl를 제공합니다. 크고 작은 마스터 플레이트도 고용 수 있습니다. 그것이 사용되지 않는 Parafilm 잘 접시를 밀봉합니다. </li></ol> 4 부 : 입술 분석 <ol><li> 폴리 프로필렌 96 - 얕은 잘 microtiter 접시는 세라를 테스트하는 데 사용됩니다. 첫 번째 단계에서, 8 채널 micropipette를 사용하여 96 - 웰 플레이트의 각 잘하기 위해 버퍼의 40 μl를 추가합니다. </li><li> 다음 마스터 플레이트에서 희석 세라 (1 μl 세라 이에 상응하는 금액)의 10 μl를 타고 8 채널 micropipette를 사용하여 작업 판의 각 물론 직접 그것을 추가합니다. </li><li> Ruc - 항원 추출물을 포함하는 마스터 믹스는 앞으로 1 X 10 7 빛의 단위 (LU)이 각각 잘하기 위해 버퍼의 50 μl에 추가됩니다 그러한 책정됩니다. 낮은 입력은 (같은 작은 X 10 6 1 등)도 사용하지만, 낮은 동적 범위의 결과하실 수 있습니다. 각 물론이 마스터 혼합 및 Ruc - 항원 혼합물의 피펫 50 μl를 확인합니다. </li><li> 실온에서 1 시간 로터리 쉐이크에 번호판을 품어. </li><li> 부화하는 동안, 새로운 96 잘 필터 HTS 접시 (Millipore, 베드 포드, MA) 각 우물의 바닥에 Ultralink 단백질 PBS에서 A / G 구슬 (피어스 생명 공학, 록퍼드, IL)의 30 % 정지 5 μl를 추가 . </li><li> 1 시간 부화 후, 단백질 8 채널 micropipette를 사용하여 A / G 비즈를 포함한 96 웰 플레이트 필터 HTS에 100 μl Ruc - 항원 항체 반응 혼합물을 전송할 수 있습니다. </li><li> 실온에서 1 시간 동안 추가 로터리 쉐이크에서 96 - 웰 플레이트 필터를 품어. </li><li> 다음 진공 매니폴드에 필터 번호판을 씻으십시오. 각 잘는 PBS의 100 μl로 두 번 다음에, 버퍼 100 μl 8 번 씻어입니다. 이것은 수동으로 또는 로봇 pipetting 워크 스테이션과 함께 수행할 수 있습니다. </li><li> 마지막 세척 후, 진공이 해제됩니다. 필터 플레이트를 제거하고 종이 타올이나 접시의 위쪽과 아래쪽에있는 수분을 제거해야하는 필터 종이 스택을 사용하여 건조 얼룩. </li></ol> 5 부 : 측정 및 데이터 분석 발광 설정 : 베르톨드의 LB 960 센트 microplate의 luminometer는 각 잘 단일 주입기를 사용하여 발광을 결정하는 데 사용됩니다. 기계가되면, 인젝터 세척주기를 사용하여 증류수 H 2 O와 주입기를 씻어. Coelenterazine 기판은 같은 제조 업체에서 설명한 Promega의 Renilla 기판 키트를 사용하여 준비가되어 있습니다. 1X coelenterazine 기판 믹스 일반적으로 6 ML (예 : 60 μl coelenterazine 주식 플러스 1X 버퍼 6 ML) 마중물 기계에 대비 한 전체 96 - 웰 플레이트를 실행합니다. 접시를 분석하기 전에 베르톨드의 LB 960 센트 microplate의 luminometer는 1X coelenterazine 기판과 함께 준비하는 것입니다. 기판을 주입하고 번호판을 읽기위한 설정을 포함하는 프로그램 파일을 엽니다. 이러한 측정, coelenterazine 기판의 50 μl를 주입, 접시는 발광의 읽기 5 초 뒤에 2 초에 대한 흥분 상태입니다. <ol><li> 플레이트 luminometer가 전체 번호판을 읽을 수있는 능력을 가지고 있지만, 접시의 일부 읽어도 (읽기 메뉴에서) 선택할 수 있습니다. 접시의 읽기 시작 프로그램을 시작합니다. </li><li> 실행 후, luminometer에 spillage을 방지하기 위해 즉시 microtiter 필터 플레이트를 제거합니다. 분석을위한 Excel 형식으로 MikroWin 프로그램 생성된 데이터를 내보낼 수 있습니다. </li><li> 우리는 두 개의 독립적인 실행에서 세라를 평가하는 것이 좋습니다. 데이터는 다음 각 샘플에 대한 LU의 titer 값을 생성하기 위해 평균입니다. 이러한 값은 더욱 버퍼 공백을 뺄 조정할 수 있습니다. </li><li> 같은 컷오프이 말은 플러스의 컨트롤 값을 3 또는 5 표준 편차를 취하여 계산 수있는 결정과 같은 추가 데이터 분석. </li></ol>

<ol>
	<li>Burbelo, P. D., Goldman, R., Mattson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunoprecipitation+method+for+quantitatively+measuring+antibody+responses+in+clinical+sera+samples+by+using+mammalian-produced+Renilla+luciferase-antigen+fusion+proteins.">A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.</a> BMC Biotechnol. 5, 22-22 (2005).</li><li>Burbelo, P. D., Leahy, H. P., Iadarola, M. J., Nutman, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Four-Antigen+Mixture+for+Rapid+Assessment+of+Onchocerca+volvulus+Infection.">A Four-Antigen Mixture for Rapid Assessment of Onchocerca volvulus Infection.</a> PLoS Negl Trop Dis. 3, e438-e438 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+luminescent+profiling+of+anti-La+and+other+autoantibodies+in+Sj+gren&#x2019;s+syndrome.">Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sj gren&#x2019;s syndrome.</a> Autoimmunity. 139, 241-245 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+antibody+quantification+and+generation+of+whole+proteome+antibody+response+profiles+using+LIPS+(luciferase+immunoprecipitation+systems).">Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems).</a> Biochem Biophys Res Commun. 352, 889-895 (2007).</li><li>Burbelo, P. D., Groot, S., Dalakas, M. C., Iadarola, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+definition+profiling+of+autoantibodies+to+glutamic+acid+decarboxylases+GAD65/GAD67+in+stiff-person+syndrome.">High definition profiling of autoantibodies to glutamic acid decarboxylases GAD65/GAD67 in stiff-person syndrome.</a> Biochem Biophys Res Commun. 366, 1-7 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+luminescence+assay+for+autoantibodies+to+mammalian+cell-prepared+insulinoma-associated+protein+2.">A new luminescence assay for autoantibodies to mammalian cell-prepared insulinoma-associated protein 2.</a> Diabetes Care. 31, 1824-1826 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+human+herpes+simplex+virus+type+1+and+2+infections+by+luciferase+immunoprecipitation+system+assay.">Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay.</a> Clin Vaccine Immunol. 16, 366-371 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+quantitative+serological+detection+of+anti-cytomegalovirus+(CMV)+antibodies.">Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies.</a> Virol J. 6, 45-45 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-antigen+mixture+containing+v-cyclin+for+serological+screening+of+human+herpesvirus+8+infection.">Four-antigen mixture containing v-cyclin for serological screening of human herpesvirus 8 infection.</a> Clin Vaccine Immunol. 16, 621-627 (2009).</li><li>Ramanathan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luciferase+immunoprecipitation+systems+assay+enhances+the+sensitivity+and+specificity+of+diagnosis+of+Strongyloides+stercoralis+infection.">A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection.</a> J Infect Dis. 198, 444-451 (2008).</li></ol>

입술은 최소한의 분석 최적화 및 그 단순 때문에, 고품질의 데이터는 일반적으로 특정 항원에 대한 이주 아래의 입술을 사용하여 생성할 수 있어야합니다. 가장 시간이 많이 걸리는 단계는 복제되고 Ruc - 항원 융합을 포함하는 적절한 플라스미드 발현 벡터를 생성. 이러한 plasmids가 생성되면 위에서 설명한 바와 같이, 하나 또는 여러 개의 항원이 빠르게 입술로 테스트할 수 있습니다. 그것은 입술은 세라도 튜브 형식이나 또는 BIOMEK 로봇 스테이션 2 또는 진공 여과와 함께 접시 세척기의 도움 microtiter 접시에 필터를 테스트 할 수있는 등 매우 다양한 것을 주목하여야한다. 이것은 확장성이 높은 시스템은 인간 autoantigens 3 또는 바이러스 4 전체 프로테옴의 패널의 프로 파일링 병렬 입술 있습니다. 그것은 입술로 생성된 다양한 항체 프로필 치료 10, 백신 모니터링 과정 다음, 진단 3-9, 항원 발견 9 유용하게 사용될 가능성이 높습니다 및 특정 질병 상태와 일반적으로 공중 보건에 대한 광범위한 의미가 있습니다.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>HiSpeed Plasmid Midi KIt</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
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<td>100 x 20 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Opti-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1814443 </td>
<td>&nbsp;</td>
<tr>
<td>Complete, Mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836153001</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 96 DeepWell 1 ml plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120 </td>
<td>Deep well plate with lid and seal with Parafilm</td>
</tr>
<tr>
<td>Polypropylene microtiter plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120</td>
<td>&nbsp;</td>
<tr>
<td>HTS Filter plate</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSBVN1B50</td>
<td>&nbsp;</td>
<tr>
<td>Ultralink Immobilized Protein A/G</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>53133 (10ml)</td>
<td>&nbsp;</td>
<tr>
<td>Renilla Luciferase Assay System</td>
<td>&nbsp;</td>
<td>Promega </td>
<td>E2820</td>
<td>For 2000 assays</td>
</tr>
<tr>
<td>Centro microplate luminometer</td>
<td>&nbsp;</td>
<td>Berthold</td>
<td>LB 960</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

antibody profiling by luciferase immunoprecipitation systems (lips)

종합적 autoantigens 및 전염성 요원 항체 프로필을 이해하고 quantifying에 대한 기술은 질병 메커니즘에 새로운 통찰력을 얻을 수 있으며 다른 임상 특징과 질환 substratify 더 나은 pathogenesis을 이해하는 새로운 마커를 명료하게하다 수 있습니다. 우리는 매우 양적 방법 감염과 관련된 autoantigens 및 병원체 항원을 환자 세라의 항체 반응을 프로 파일링에 대한 루시페라제 Immunoprecipitation 시스템 (입술)라는 개발했습니다. ELISAs 달리, 매우 민감한 입술은 쉽게 보편적인 형식으로 다른 항원에 체액 serological 응답 프로파일을 조사하기 위해 구현 및 동적 항체 titer 6 로그온하는 최대 범위 생산합니다<sub&gt; 10</sub&gt; 몇 가지 항원에 대한. 이러한 연구에서는 항원의 패널 또는 일부 병원균 (즉, HIV)의 전체 프로테옴에 대한 환자의 체액 반응의 입술로 양적 프로파일은 일반적으로 엘리사에 의해 하나의 항원 테스트를보다 유익한 것입니다. 또한, 입술은 또한 시간과 높은 정화 항원뿐만 아니라 표준 ELISAs에 필요한 노동 집약적인 분석 최적화 단계를 생산하는 데 필요한 노력을 제거합니다. 우리가 쉽게 하나 또는 여러 항원에 항체 반응을 프로 파일링을위한 입술 assays를 수행의 기술적 측면을 설명하는 자세한 절차를 제공합니다.

Watch this Scientific Journal Video about Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS at JoVE.com

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

입술 (루시페라제 Immunoprecipitation 시스템)를 수행하는 기술적인 측면이 설명되어 있습니다. 전체 접근 방식은 포유 동물 세포에서 Renilla 루시페라제 (Ruc)에 융합 항원을 인코딩 키메라 유전자 표현이 포함됩니다. 원유 Ruc - 항원 추출물 다음 준비하고, 정화없이 항체를 정할 immunoprecipitation의 assays에 근무하고 있습니다.

루시페라제 Immunoprecipitation 시스템 (입술)에 의해 항체 프로파일

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

antibody-profiling-by-luciferase-immunoprecipitation-systems-lips

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JoVE Journal

Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

26.7K 조회수.  National Institute of Dental and Craniofacial Research, National Institutes of Health. 입술 (루시페라제 Immunoprecipitation 시스템)를 수행하는 기술적인 측면이 설명되어 있습니다. 전체 접근 방식은 포유 동물 세포에서 Renilla 루시페라제 (Ruc)에 융합 항원을 인코딩 키메라 유전자 표현이 포함됩니다. 원유 Ruc - 항원 추출물 다음 준비하고, 정화없이 항체를 정할 immunoprecipitation의 assays에 근무하고 있습니다.

비디오: Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

Microelectrode 구조에 의해 발생 AC 계면 현상

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

연구

생물학

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Methylated DNA의 Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM : 유동세포계측법에 의해 감지 Immunoprecipitation

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

단지 샘플의 특정 단백질 글리코 실화의 다중 높은 처리량 프로파일을위한 화학을 차단 항체 Microarray

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Polysom​​e 프로파일에 의한 스트레스 조건에서 번역 개시 분석

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

정량적 RT-PCR에 의해 개별 인간 배아 줄기 세포를 프로파일 링

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polysom​​e 프로파일 링 포유 동물 세포에 선택적 mRNA의 번역 평가

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...