우리는 덕분에 금융 지원을 위해 다음과 같은 것이다 : BBSRC 연구소 전략 프로그램 기금 - CF 및 TMW을; BBSRC 동부 바이오 DTP 자금 - LG; 에딘버러의 다윈 신뢰 - MLH. 우리는 또한이 원고의 TREM2 최적화를 포함 할 수있는 권한에 대한 박사 배리 맥콜에게 감사의 말씀을 전합니다.

<ol>
	<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Biotechnology. 1992 (24), 145-149 (1979).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Protocols on CD-ROM. , (1998).</li><li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+immunodetection.">Immunoblotting and immunodetection.</a> Curr Protoc Mol Biol. Chapter. 10, 10 (2008).</li><li>Eaton, S. L., 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=Total+protein+analysis+as+a+reliable+loading+control+for+quantitative+fluorescent+Western+blotting.">Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.</a> PLoS One. 8 (8), 72457 (2013).</li><li>Suzuki, O., Koura, M., Noguchi, Y., Uchio-Yamada, K., Matsuda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sample+mixtures+for+standard+curve+creation+in+quantitative+Western+blots.">Use of sample mixtures for standard curve creation in quantitative Western blots.</a> Exp. Anim. 60, 193-196 (2011).</li><li>Colella, A. D., 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=Comparison+of+Stain-Free+gels+with+traditional+immunoblot+loading+control+methodology.">Comparison of Stain-Free gels with traditional immunoblot loading control methodology.</a> Anal Biochem. 430 (2), 108-110 (2012).</li><li>Zellner, M., 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=Fluorescence-based+Western+blotting+for+quantitation+of+protein+biomarkers+in+clinical+samples.">Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.</a> Electrophoresis. 29, 3621-3627 (2008).</li><li>Kielar, C., 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=Molecular+correlates+of+axonal+and+synaptic+pathology+in+mouse+models+of+Batten+disease.">Molecular correlates of axonal and synaptic pathology in mouse models of Batten disease.</a> Hum Mol Genet. 18, 4066-4080 (2009).</li><li>Mutsaers, C. A., Lamont, D. J., Hunter, G., Wishart, T. M., Gillingwater, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+proteomics+identifies+Calreticulin+and+GRP75/Mortalin+as+peripherally+accessible+protein+biomarkers+for+spinal+muscular+atrophy.">Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy.</a> Genome Med. 5 (10), 95 (2013).</li><li>Wishart, T. M., 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=Dysregulation+of+ubiquitin+homeostasis+and+&#946;-catenin+signaling+promote+spinal+muscular+atrophy.">Dysregulation of ubiquitin homeostasis and &#946;-catenin signaling promote spinal muscular atrophy.</a> J Clin Invest. 124 (4), 1821-1834 .</li><li>Mathews, S. T., Plaisance, E. P., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+systems+for+westerns:+chemiluminescence+vs.+infrared+detection.">Imaging systems for westerns: chemiluminescence vs. infrared detection.</a> Methods Mol Biol. 536, 499-513 (2009).</li><li>Hawes, J. J., Brunzell, D. H., Wynick, D., Zachariou, V., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GalR1,+but+not+GalR2+or+GalR3,+levels+are+regulated+by+galanin+signalling+in+the+locus+coeruleus+through+a+cyclic+AMP-dependent+mechanism.">GalR1, but not GalR2 or GalR3, levels are regulated by galanin signalling in the locus coeruleus through a cyclic AMP-dependent mechanism.</a> J Neurochem. 93 (5), 1168-1176 (2006).</li><li>Bond, D., Primrose, D. A., Foley, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+signaling+events+in+Drosophila+S2+cells.">Quantitative evaluation of signaling events in Drosophila S2 cells.</a> Biol Proceed Online. 10 (1), 20-28 (2008).</li><li>Wishart, T. M., 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=Differential+proteomics+analysis+of+synaptic+proteins+identifies+potential+cellular+targets+and+protein+mediators+of+synaptic+neuroprotection+conferred+by+the+slow+Wallerian+degeneration+(Wlds)+gene.">Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene.</a> Mol Cell Proteomics. 6, 1318-1330 (2007).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fastest+western+in+town:+a+contemporary+twist+on+the+classic+western+blot+analysis.">The fastest western in town: a contemporary twist on the classic western blot analysis.</a> J. Vis. Exp. (84), 51149 (2014).</li></ol>

The authors have nothing to disclose.

등을 고려해서 계획은 실험에 앞서 필수적이며 궁극적으로 사용 된 기술의 성공 여부를 확인할 수 있습니다. 적절한 항체, 전송 및 시각화 방법을 사용하기로 선택하려고 할 때 잠재적 인 장애물의 과다를 제공 할 수 WB를 사용하여 단백질 검출 발전. 다행히, 단백질의 존재와 샘플 사이 점점 더 미묘한 표현의 차이를 결정하기 위해 일상적으로 사용할 수있는 QFWB주의 깊게 점검하고 적절한 통제 수단을 사용. 이 프로토콜은 방지 및 / 또는 그와 관련된 많은 일반적인 함정 중 일부를 극복하기 위해 형광 양적 서부 모래 바닥뿐만 아니라 몇 가지 문제 해결 전략에 포괄적 인 가이드를 제공합니다. 감도를 유지하고 획득하기 위해 사용되는 중요한 단계는 진정으로 정량화 및 비교 측정은 다음과 같습니다 : 조직 샘플에서 1) 강력한 단백질 추출; 2) 샘플 준비; 3) 정확한 단백질 loading는 총 단백질 분석에 의해 측정; I-의 얼룩을 사용하여 단백질의 4) 최적의 전송; 열전달 및 관련 소프트웨어를 사용하여 0.1 % 트윈 20을 포함하는 버퍼를 차단하는 일차 및 이차 항체의 5) 준비, 6) 올바른 시각화 및 분석. 적외선 형광 검출은 진정으로 정량적이고 전통적인 ECL 검출 기법 3,11에 비해 민감도를 제공한다. 이 검출 시스템은 여러면 처리이며, 같은 QFWB에 한정되지 않는다. 이 시스템은 전체 조직 절편 (12)의 시각화 및 정량화를 허용 저전력 면역 조직 라벨링 촬상 할 수있다. 이 정량적 평가의 측면에서 기존의 현미경과 기존의 면역 캡처 필적까지 빨간색 영상을 볼 수 있었다 해상도의 측면에서 잠재적 인 미래 개발의 한 영역이다. 그러나, P의 미묘한 변화에 대한 민감도와최소 제어 조치를 유지 표현이 변동성을 보장하기 위해 매우 중요합니다 rotein하는 강력한 프로토콜과 엄격한이다. 이 샘플 로딩이 균일 검색이 현실과 관련하여 제조업체의 지침을 테스트하는 것은 전송하는 것입니다 여부를 결정하기 위해 기본 및 보조 항체의 최적화임을 보증을 제공하기 위해 총 단백질 스테인드 젤의 생산 다음에 조직 샘플에서 엄격한 단백질 추출로 시작 시간 효율적인 단백질 전달을 얻었다. 그럼에도 불구하고, WB의 조건이 최적화되는 경우에도, 여전히 문제가 완전히 여기에 탐험되지 않았을 수 있습니다 서부 실행과 관련이있다. 이들은 다음을 포함하지만, 단백질의 가용화 및 추출 완충액의 선택 등 다양한 요인에 한정되지 않는다. 일부 버퍼 단백질 농도 분석을 방해 할 수 있지만, 몇몇 조직은 자동화 macera의 사용으로 더 강력한 기술을 필요로 용해하는 것이 특히 어렵다팅은 맥의 dissociator과 함께 M 튜브 등의 용기를 밀봉. 또한, -80 ° C에서 추출 및 비 추출 된 물질의 저장을위한 간단한 제어 조치를 즉시 추출 후 최적의 라벨을 획득하고 가난한 결과 주 후 가진 사이의 차이가 될 수 있습니다. 현대 QFWB 방법은 단백질 발현에 미묘한 차이를 캡처 더 민감한 것으로 입증 및 ECL 같은 이전의 기술과 비교했을 때 더 융통성 동시 듀얼 라벨 3을 허용한다. 그것은 서양 블로 팅 프로토콜이 강력하고 정확한 정량 및 통계 분석을 위해 쉽게 반복적 것이 중요합니다. 이 프로토콜은 서로 다른 조직 샘플 종 (3)의 종류에 걸쳐 단백질의 검출을 위해 일상적으로 사용하기에 민감하고 강력한 충분히 따라서 실험 1 당 소비 사용량뿐만 아니라 시간을 감소 동일한 QFWB 내 로우 및 하이 풍부 단백질의 정량화를 허용. 9,14 그러나 정확성함으로써 잘못된 데이터 수집을 방지하기 위해 준수해야 중요하고 적절한 통제 조치의 포함이다 오믹 연구 - (1) 또한,이 기술의 증가 감도는 점점 더 인기를 검증 할 수 있습니다.

웨스턴 블롯 (WB)는 원래 같은 조직 파쇄 한 바와 같이, 복잡한 생물학적 샘플에서 목적 단백질의 존재 여부를 결정하기 위해 1970 년대 후반에 개발 된 분석 기술이다. 일반적으로 인해 키 항체 - 항원 상호 작용, 단백질 면역라고, 방법론은 5 별개의 단계로 구성되어 자신의 등전점에 의한 단백질의 1) 전기 영동 분리; 2) 니트로 셀룰로오스 또는 폴리 비닐 리덴 디 플루오 라이드 (PVDF) 멤브레인로 전송; 3) 관심 단백질에 특이 차 항체를 사용하여 라벨링; 차 항체에 대항 차 항체 4) 배양; 5) 시각화. 시각화 방법론은 안전과 감도를 개선하기 위해 시간이 진화하고있다. 첫 번째 WBS의 일부는 더 널리 사용 chemilluminescent (ECL) 방법 색채와에 진행 라디오 라벨 태그를 사용하여 수행되었다. Radioactivi비색과 ECL 방법은 알칼리 포스 파타 아제 또는 스트렙 타비 딘 고추 냉이 과산화 효소 (HRP) 2 등의 효소 기자와 간접 라벨링 기술을 사용하는 반면 타이 직접 특정 항원에 대한 프로브에 표지 하였다. 크로마 또는 발광 제품 라벨의 강도가 강하거나 약한 신호 강도는, 샘플에 관심 단백질의 다소간 존재를 나타내고있다 농도계를 사용하여 측정한다. ECL은 더욱 민감하고, 따라서이 방법론 선호되지만 3 방법은 초기에이어서 3 확립보다 정교한 디지털 이미징 기술로 엑스레이 필름이 개발되었다. WB의 디지털 이미징의 발전뿐만 아니라 그들의 관심 단백질의 존재 여부를 결정하기 위해 연구를 허용하지만, 다른 샘플과 비교할 때, 또한 선택된 단백질의 발현 수준에 대한 추론을 허용하고, 따라서로 지칭 될 수있다 &quot;반 정량적 &amp; #8221 ;. 측정 된 형광의 레벨을 직접 시료 내의 수량 및 단일 단백질의 발현과 관련된다 최근 진정한 정량적 더 민감 웨스턴 블럿 기술이 개발되었다 : 정량적 (QFWB) 웨스턴 블로 팅 형광등. ECL 표지와 QFWB를 비교하면, 형광 이차 항체의 사용은 선형 검출 프로파일 (4)를 생성한다. 이것은 신호 선형성 일반적 도처 발현 하우스 키핑 유전자 5,6로, 즉, 직접 단백질 발현과 관련된 5 μg의 신호 포화 아래 낮은 단백질 부하 발생 ECL 기법 대조적이다. 이 불균형은 대부분 전위 신호 포화의 높은 가능성의 결과로, 보조 바이오틴에 결합하는 아비딘 ECL 기판에 사용할 결합 부위의 수에 의해 크게 유발된다. 이것은 같은 ONL 지칭되는 ECL베이스 면역의 주된 이유 중 하나이다Y &quot;반 정량적&quot;7. 발현 수준에서의 미묘한 차이를 측정하고 측정에 부정확을 초래할 수있는 경우 신호의 포화 점은 매우 중요하다. 최근 몇 년 동안 계속 증가하는 민감도와 미묘한 표현 차이의 식별을 자세히 널리 프로테오믹스 기술의 출현은 검증 실험 8,9에 대한 진정한 양적 웨스턴 블로 팅에 계속 증가하는 의존 가져왔다. 민감한 강력한 진정 정량적 방법론의 적용은 따라서 중요하다. 많은 &quot;최적화&quot;서양 블롯 방법은 자주 인해 공식적인 문서화 된 프로토콜에서 쉽게 보이지는 미묘한 방법론 조정을 설정하거나 복제하기 어려울 독립적 인 실험실에 의해 이용되어왔다. 이 QFWB에 설립하고 강력한 프로토콜이며 추가로 일반적인 문제 그쪽으로 문제 해결을위한 유용한 전략을 제공합니다T 구현시 발생할 수 있습니다. 이 프로토콜은 원래 쥐의 뇌 균질 물과 함께 사용하기 위해 최적화되어 있지만, 이후 조직 샘플과 종 4,9,10의 넓은 범위에 걸쳐 효과적으로 사용되어왔다. 특정 문제를 해결하기 위해 필요한 잠재적 인 프로토콜의 변화가 포함되어 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:427px;">Name of Material/ Equipment</td>
			<td style="width:255px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:224px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">RIPA Buffer</td>
			<td style="width:255px;">Fisher Scientific UK Ltd</td>
			<td>10230544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">M Tubes</td>
			<td style="width:255px;">Miltenyi Biotec Inc.</td>
			<td>130-093-236</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:427px;">iBlot Transfer Stack, PVDF Regular</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>IB401001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MagicMark XP Western Protein Standard (20-220 kDa)</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>LC5602</td>
			<td>Use in gel 1 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">SeeBlue Pre-stained protein standard</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>LC5625&#160;</td>
			<td>Use in gel 2 Total protein stained gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NuPAge LDS Sample buffer 4X</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:427px;">NuPAGE MES SDS Running Buffer (for Bis-Tris Gels only) (20X)</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 12 well</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:427px;">PHOSPHATE BUFFERED SALINE TABLET,*TRU-ME, PHOSPHATE BUFFERED SALINE TABLET</td>
			<td style="width:255px;">Sigma-Aldrich, UK</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Micro BCA, Protein Assay Kit</td>
			<td style="width:255px;">Fisher Scientific UK Ltd</td>
			<td>10249133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Odyssey&#160; blocking buffer</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td>P/N 927-40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 680RD Goat anti-Rabbit IgG (H+L), 0.5 mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-68071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 680RD Donkey anti-Mouse IgG (H+L), 0.5 mg&#160;</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-68072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 800CW Goat anti-Rabbit IgG (H + L), 0.5mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-32211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 800CW Donkey anti-Goat IgG (H + L), 0.5mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-32214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">ODYSSEY CL Infra-red imager</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;"></td>
			<td>Call for quotation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">iBlot 7-Minute Blotting System</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td style="width:119px;"></td>
			<td>This model is no longer in production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">InstantBlue Protein stain</td>
			<td style="width:255px;">Expedeon, UK</td>
			<td>ISB1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Revitablot western blot stripping buffer&#160;</td>
			<td>Rockland Immunochemicals Inc.</td>
			<td>MB-085-0050</td>
			<td></td>
		</tr>
	</tbody>
</table>

a guide to modern quantitative fluorescent western blotting with troubleshooting strategies

1970 년대 말은 웨스턴 블롯, 존재와 복잡한 생체 시료 내 특정 단백질의 상대적인 풍요 로움을 평가하는 기술의 첫 번째 공식보고 사용을 보았다. 그 이후로, 웨스턴 블로 팅 방법은 분자 생물학 실험적인 레퍼토리의 공통 요소가있다. 용어는 &quot;웨스턴 블롯&quot;을 사용하여 PubMed에의 피상적 검색 이십이만 출판 원고를 초과 중요한 것은 2014 년에이 기술의 사용을 만든 것을 제안, 지난 10 년은 개발과 함께 기술 이미징의 발전을 보았다 감도 개선 및 선형 검출 더 큰 범위를 산출 한 민감한 형광 라벨. 결과는 생물 학자들이 이전보다 민감도와 정확도를 비교 발현 분석을 수행 할 수 있습니다 웨스턴 블롯 (Western Blot) (QFWB)을 기반으로 지금 진정으로 계량화 형광입니다. 많은 &quot;최적화&quot;서양 블롯방법론이 존재하고 서로 다른 실험실에서 사용된다. 이들은 종종 인해 미묘하지만 문서화되지 않은 절차 개정의 요구에 구현하기 어려울. 이 프로토콜은 문제 해결 전략을 완성 설립하고 강력한 QFWB 방법의 포괄적 인 설명을 제공합니다.

QFWB 감도 및 검출의 선형 범위를 기존의 ECL 검출보다 큰으로하여 효율적인 해석을 돕는 정확한 데이터가 수집되는 것을 보장하기 위해 매우 중요하다 통제 조치의 숫자가 있습니다. 첫째로, 양성 대조군 샘플의 포함은도 1에 도시 된 바와 같이. 둘째, 전송 최적화 멤브레인 겔로부터 고 분자량 및 저 분자량 단백질의 상응하는 이동을 보장하기 위하여도 2에 나타나는 바와 같이. 셋째, 항체의 최적화, 특히 이차 그 최적화 종종 간과되지만, 항체는 관심 단백질 (들)의 정확한 해석을 방해 할 밴딩 비특이적 생산할 수있다. 도 3 참조. 넷째, 또한 수있다 경우 단백질이 검출되지 나타나지만 존재할 것으로 예상되는 경우, 이는 단순히 보조 RAIS를 사용하여 보정 될 수 이차 항체 문제가 될 수 있다는다른 종의 호스트에 에드. 도 4를 참조. 다섯째, 총 단백질 라벨 및 분석 도처 내부 참조 표준 3 표현 전통적인 단일 단백질 (들)의 사용에 비해 훨씬 더 견고하고 정량화 방법이다. 이러한 단일 단백질의 대부분은 차동 퇴행성 신경 질환의 모델 간에는 물론 다른 조직 샘플에서 발현되는 것으로 밝혀졌다 표현의 균일 성이 동일한 조직 내 3 변경할 수있다. 표준 오차를 나타내는 비교하고, 각 샘플에 대해 측정 된 다양한 분자량 범위에서 각 레인에서 단백질 부하를 정량함으로써 총 단백질 분석과 결합 될 때,도 6에서 입증 된 바와 같이 따라서로드 제어 겔의 제조는 시료 하중의 균일 성을 확인한다 . 중요한 것은, 이러한 문제 해결 기술 및 모든 컨트롤 만 분석 t의 민감도 일관성만큼 효과적운영자 (그림 5)에 의해 적용 ools. 마지막으로이 기술은 스트리핑과 같은 요인을 포함하지만 장기간 저장 조건 하에서 감도 증가, 감소 된 배경, 이중 색상 검출 및 막 안정성에 한정되지으로 ECL보다 더 유연성을 가진 멤브레인의 재 프로빙라는 것으로. 그림 7을 참조하십시오.

Watch this Scientific Journal Video about A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies at JoVE.com

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

문제 해결 전략 현대 양적 형광 웨스턴 블로 팅에 가이드

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

a-guide-to-modern-quantitative-fluorescent-western-blotting-with

Research

JoVE Journal

Biology

50.7K 조회수.  University of Edinburgh. The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

비디오: A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

웨스턴 Invitrogen NuPage Novex BIS 트리스 MiniGels를 사용하여 모래 바닥

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

연구

생물학

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

유전자 Inducible의 운명 매핑에 대한 실용적인 접근법 : 셀을 선택하고 추적하는 비주얼 가이드 VIVO에서</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

웨스턴 모래 바닥 : 샘플 준비를 탐지하기 위해

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

중합 효소 사슬 반응 : 기본 프로토콜 플러스 문제 해결 및 최적화 전략

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

서부 블로팅에서 실용적이고 편리하며 신뢰할 수 있는 토탈 단백질 로딩 제어를 위한 V3 얼룩 없는 워크플로우

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

비전문가를위한 계통 발생에 대한 실용 가이드

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

타운 웨스턴 가장 빠른 : 클래식 웨스턴 블롯 분석에 현대 트위스트

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

아마의 꽃 - 딥 변환 (<em&gt; LINUM usitatissimum</em&gt;) 높은 변환 속도와 형질 전환 자손을 생성합니다

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

멀티 플렉스 애플리케이션을위한 형광 단백질 유전자 바코드

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

내부 양성 대조군과 양적 재조합 효소 중합 효소 증폭 분석 개발

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...