이 작품, Fondo 나시오날 드 개발 Científico y Tecnológico (Fondecyt), 칠레, 프로젝트 11130263 (CW)에, NERC 프로젝트 CONICYT + 프로그램이 드에 의해 지원 되었다 (CW)에 Colaboración 국제 PCI-PII20150073 및 Vicerrectoría de에서 U-inicia Investigación 대학 데 칠레 (CW)입니다.
Ciencias Químicas y를 박사 호르헤 Babul 그리고 디에고 Quiroga-로저 유용한 토론, 원고 및 주소 드 extensión 드 라 Facultad 드 수정에 대 한 로버트 Lesch에 대 한이 프로젝트의 초기 단계에 있는 도움에 대 한 타 호텔 팔 마에 감사 합니다. 칠레 대학에서에서 Farmacéuticas입니다. 마르셀 페 레 스는 비디오 편집을 제공 하는 음성-텍스트 및에 롤 왓슨 해결을 위한 닐 스티븐 스 너무 감사.

1. 준비 하 고 실행 하는 젤
<ol><li>비 변성 시키기 12 %polyacrylamide 젤 젤 Sambrook와 러셀14 에 찾았지만 SDS 대신 물을 사용 하 여 나트륨 라우릴 황산 Polyacrylamide 젤 전기 영동 (SDS-PAGE) 제조 법을 사용 하 여 준비 합니다.<ol>
<li>젤을 해결의 5 mL를 준비 하려면 1.7 mL 증류수, 아크릴 아 미드/비스 아크릴 아 미드 (30/0.8 %w / v)의 2 개 mL, 1.5 M Tris pH 8.8의 1.3 mL, 10% 염화 persulfate의 0.05 mL 및 0.002 mL TEMED의 사용 합니다.</li>
		<li>준비의 겹쳐 쌓이는 젤 2 mL 물 1.5 mL, 아크릴 아 미드/비스 아크릴 아 미드 (30/0.8 %w / v)의 0.33 mL, 1 M Tris pH 6.8의 0.25 mL, 10% 염화 persulfate, 0.02 mL 및 0.002 mL TEMED의 사용 합니다. 젤의 크기는 7.3 c m x 8.2 c m 0.075 x cm (l x w x d). 참고: 0.75 m m 두께의 15 잘 빗을 사용 하 여 최고의 감도 대 한.</li>
	</ol>
</li>
	<li>버퍼 및 1.5 mL microcentrifuge 튜브 (6 x 로딩 버퍼 DNA 샘플의 5 µ L의예: 사용 1 µ L)에 DNA 샘플 로드 x 6의 적절 한 금액을 추가 합니다.</li>
	<li>수직 전기 영동 챔버에 젤을 놓습니다.<ol>
<li>2 젤을 실행 1 x TAE 버퍼의 750 mL 챔버를 채우십시오. 50의 1 리터를 준비 하기 x TAE, 트리 스 기지의 442 g, 빙 초 산의 57.1 mL 및 100 mL의 0.5 M EDTA pH 8 사용 하 여. 증류수 1 l.의 최종 볼륨을 추가</li>
		<li>젤에서 샘플을 로드 합니다. 100 V 2 h에 젤을 실행 합니다.</li>
	</ol>
</li>
	<li>또는, Sambrook14에서 제조 법을 사용 하 여 1 x TAE agarose 젤 준비.<ol>
<li>1의 300 mL를 추가 수평 전기 이동 법 약 실에 x TAE. 100 V에서 45 분 젤을 실행 합니다.</li>
	</ol>
</li>
</ol>2. NBT SG 나 얼룩 (그림 1).
<ol><li>
아크릴 아 미드 젤 얼룩입니다.
	<ol>
<li>솔루션을 준비: 0.1 %w / v NBT 및 1.2 SG x 나. 참고: 그것은 ethidium 평범한 사람, GelRed 그리고 SYBR 골드 SG 대신 사용할 수, 하지만 SG 최상의 성능을 했다.</li>
		<li>1.2의 16.75 mL를 붓고 x SYBR 그린 나 적절 한 크기의 용기에 솔루션. 참고: 우리는 컨테이너, 피 펫 팁 상자 뚜껑을 사용 하 고 이러한 볼륨 커버 젤;이 컨테이너를 사용 하 여 계산 됩니다. 그것의 차원은 11.6 cm x 7.6 cm x 2.6 c m (w x d x l).</li>
		<li>솔루션에는 젤을 놓습니다. 0.2 m m NBT 주고 0.1 %w / v NBT 솔루션의 3.25 mL을 추가 및 1 x (2.0 µ M에 해당) SG 나 최종 농도.</li>
		<li>플라스틱 필름, 컨테이너를 봉인 하 고 알루미늄 호 일로 어떤 주변광을 차단 하도록 컨테이너를 완전히 포장. 참고: 플라스틱 필름 튀는 방지 하 고 얼룩 솔루션으로 알루미늄 호 일의 반응을 방지 하는 데 사용 됩니다.</li>
		<li>25 분 60 rpm에서 궤도 셰이 커에 흔들어.</li>
		<li>알루미늄과 플라스틱 커버를 제거 합니다. 최대 90 분 동안 햇빛에 노출. 관심의 밴드 표시 될 때까지 5 분 마다 확인 합니다.</li>
		<li>푸른 빛을 사용 하려면 3 발광 다이오드 (LED)와 protoboard를 준비 하 고 젤 표면 (표, 시간으로 얼룩 LED 광원의 강도에 따라 달라 집니다 필요)에서 약 5 m m Led를 배치 합니다.</li>
	</ol>
</li>
	<li>
Agarose 젤 얼룩입니다.
	<ol>
<li>준비 하 고 실행 하는 1 %agarose 젤 1.4 섹션에 설명 된 대로 후 (크기는 1 cm x 6.2 cm x 7 cm; l x w x d), 이러한 수정 2.1에서 동일한 단계를 수행:</li>
		<li>1.2의 41.9 mL를 붓고 SG x 나 피 펫 팁 상자;의 뚜껑으로 그것의 차원은 11.6 cm x 7.6 cm x 2.6 c m (w x d x l). 0.1 %NBT 솔루션의 8.1 mL를 추가 합니다.</li>
		<li>플라스틱 필름, 컨테이너를 봉인 하 고 알루미늄 호 일로 어떤 주변광을 차단 하도록 컨테이너를 완전히 포장.</li>
		<li>1 h 60 rpm에서 궤도 셰이 커에 흔들어.</li>
	</ol>
</li>
</ol>3. 데이터 분석입니다.
<ol><li>
NBT SG 나 보정 곡선.
	<ol>
<li>1200 dpi에서 스캔 하 여 젤 이미지를 얻을.</li>
		<li>이미지 분석을 위한 오픈 소프트웨어입니다. 이미지 편집기를 사용 하 여 이미지 직선 화. 이미지 densitometry를 실행 합니다.</li>
		<li>곡선 아래 면적을 계산 합니다. DNA 농도 대 신호/신호최대 의 그래프를 준비 합니다.</li>
	</ol>
</li>
</ol>4. DNA 복구입니다.
<ol><li>1에 1 %agarose 젤 준비 제조 법을 사용 하 여 x TAE Sambrook14에서 발견.</li>
	<li>1의 300 mL를 추가 수평 전기 이동 법 약 실에 x TAE. 100 ng / µ L pDJ10015 플라스 미드 DNA 사다리 구조 그림 2에 다음의 4 µ L을 로드 합니다. 100 V에 1 시간을 위한 젤을 실행 합니다.</li>
	<li>표시와 두 2 mL microcentrifuge 튜브 무게.</li>
	<li>젤을 두 개의 동일한 반쪽 위해서는 중간에 삭감. 각은 사다리 및 다른 얼룩 기법 비교 샘플 플라스 미드를 포함 해야 합니다. 그림 2에 도식 적인 표현이입니다.</li>
	<li>얼룩 하나 절반 NBT SG 나 2 단계에서.<ol>
<li>깨끗 한 메스를 사용 하 여 플라스 미드 밴드를 잘라. 젤 조각 한 튜브에 저장 하 고 무게.</li>
	</ol>
</li>
	<li>SG와 젤의 다른 부분에 얼룩이 나 1의 50 mL와 함께 SG x 나 솔루션 천천히 어둠 속에서 (60 rpm) 1 시간을 위한 젤을 흔들어.<ol>
<li>transilluminator에 젤을 놓고 깨끗 한 메스를 사용 하 여 플라스 미드 밴드 2 분 컷에 대 한 노출 합니다.</li>
		<li>다른 튜브에 젤 조각을 저장 하 고 무게. 참고: 프로토콜 일시 중지할 수 있습니다 여기-20 ° c.에 튜브를 유지 하면서</li>
	</ol>
</li>
	<li>플라스 미드 추출 상업 젤 추출 키트를 사용 하 여 수행 합니다.</li>
</ol>5. 플라스 미드 정규화입니다.
<ol><li>1.5 mL microcentrifuge 튜브에서 추출 된 플라스 미드의 10 µ L와 내가 물 형식의 60 µ L를 추가 합니다. 믹스 60 µ L 석 영 cuvette에 전송 하거나는 Nanodrop에서 직접 2 µ L를 사용 하 여.</li>
	<li>230 320 사이 흡수 스펙트럼을 측정 nm. 다음 수식을 사용 하 여 샘플의 플라스 미드 농도 계산. <img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg"> 여기서 D 는 적용 희석 요소 (이 경우에 그것은 70/10), Ax 는 x nm에서 흡수 하 고 50 µ g/ml dsDNA에 대 한 변환 인수입니다.</li>
	<li>9.1 ng / µ L를 희석 샘플입니다.</li>
	<li>대장균 (대장균) 유능한 세포 (예: XL10 금) 변환 다음 희석 샘플의 5 µ L를 사용 하 여 이노우에 변환 절차16 .</li>
	<li>배양 접시에 식민지의 수를 계산 하 고 희석 합니다. 다음 수식17:: 콜로 니 형성 단위 (CFU) 계산 <img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg"> 여기서 D 는 초기 문화에서 적용 되는 희석 비율입니다.</li>
	<li>짝이 없는 t-검정을 수행 합니다.</li>
</ol>

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저자는 공개 없다.

소설과 민감한 DNA 방법 얼룩 NBT의 감소 및이 문서에서 제시 했다 SG의 사용에 따라. 이 프로토콜에서 중요 한 단계는 NBT SG에 젤의 부 화 시간이 나 솔루션 및 NBT의 농도. Agarose 젤에 대 한 착 시간 agarose 젤 큰 두께 때문에 아크릴 아 미드 젤에 대 한 이상 이다. 이 메서드는 NBT 그것 접촉 침전으로 SDS 존재 잘 작동 하지 않습니다. 젤 보라색 배경 얻습니다, 경우 다른 젤 준비는 것이 좋습니다 및 빛의 ?...

생화학과 분자 생물학의 발전, DNA와 관련 된 연구는 DNA 분석을 더 나은 기술을 요구 했다. DNA 얼룩에 대 한 첫 번째 방법은 실버 얼룩, 매우 민감한 하지만 부족 선택 이었고 샘플 복구를 허용 하지 않습니다. 나중에, 형광 DNA 얼룩의 개발 샘플 복구의 가능성과 함께 DNA의 선택적 정량화를 허용. 첫 번째 형광 염료 DNA 정량화 사용 중 하나는 돌연변이2ethidium 평범한 사람1, 이었다.입니다. 그러나, 이제는 안전 하 고 더 민감한, GelRed, SYBR 녹색 등 향상 된 형광 염료 나 (SG 나)3; 그러나 모두 이러한 형광 염료의 자외선 (UV) transilluminator 또는 fluorimeter의 사용을 요구 한다.
가시 메 틸 렌 블루4 등 크리스탈 바이올렛5,6,7,,89, DNA, 얼룩에 대 한 다른 기술이 있다 하지만 이러한 모든 감소 된 감도에서 고통 및 선택도입니다. Tetrazolium 소금은 유기 화합물 감소, 취약 하 고이 경우 그들은 형성 된 불용 성 색된 formazan 침전10,11. 최근, 일부가 tetrazolium 소금 그들의 긍정적인 tetrazolium 반지12때문에 DNA에 바인딩할 표시 되었습니다.
최근 게시13, 얼룩 polyacrylamide 젤에서 DNA를 계량 하는 새로운 표시 기술 제안 했다, 니트로 블루 tetrazolium (NBT) 불리고 ethidium 평범한 사람, GelRed, 같은 형광 염료가 tetrazolium 소금의 감소를 사용 하 여 SYBR 녹색 어 SYBR 골드. 푸른 빛 또는 햇빛 있을 때 일을 하 고 향상 된 품질로 샘플 복구 허용이 반응에 비해 SG 사용 하 여 나 UV transilluminator와 함께. 이 문서의 목적은 tetrazolium를 사용 하 여 착 색 기술의 상세한 프로토콜 제공 하 염입니다.

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Nitro blue tetrazolium</td>
			<td style="width:227px;">Gold Biotechnology</td>
			<td style="width:307px;">298-83-9</td>
			<td style="width:64px;">reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">SYBR Green I 10000X</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">S7563</td>
			<td>reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Gel extraction kit</td>
			<td style="width:227px;">QIAGEN</td>
			<td style="width:307px;">28704</td>
			<td>used to extract plasmid from the gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">DNA ladder</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">SM 1331</td>
			<td>used to make calibration curves and as reference to cut band in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Agar Agar</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;"></td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">sodium chloride</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1064045000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">yeast extract</td>
			<td style="width:227px;">Becton, Dickinson and Company</td>
			<td style="width:307px;">212750</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">peptone</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">72161000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">TEMED</td>
			<td style="width:227px;">AMRESCO</td>
			<td style="width:307px;">761</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">ammonium persulfate</td>
			<td style="width:227px;">Calbiochem</td>
			<td style="width:307px;">2310</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">acrylamide</td>
			<td style="width:227px;">invitrogen</td>
			<td style="width:307px;">15512-023</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">bisacrylamide</td>
			<td style="width:227px;">SIGMA</td>
			<td style="width:307px;">146072</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Tris-base</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1083821000</td>
			<td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">EDTA</td>
			<td style="width:227px;">J.T. Baker</td>
			<td style="width:307px;">8993-01</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Acetic acid</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1,000,632,500</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">agarose</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">16802</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">spectrophotometer</td>
			<td style="width:227px;">Tecan</td>
			<td style="width:307px;">infinite 200 pro</td>
			<td>used to quantify DNA plasmid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">water purificatiom unit</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">Elix 100</td>
			<td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">distilled water</td>
			<td style="width:227px;"></td>
			<td style="width:307px;">-</td>
			<td>used in gels, culture medium, stainings and general solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">HCl</td>
			<td style="width:227px;">J.T. Baker</td>
			<td>9535-03</td>
			<td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">6X DNA loading dye</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">R0610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">5 mm Blue LED</td>
			<td style="width:227px;">Jinyuan electronics</td>
			<td style="width:307px;">SP-021</td>
			<td>light source used in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">protoboard</td>
			<td style="width:227px;">KANDH</td>
			<td style="width:307px;">KH102</td>
			<td>light source support and circuit&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">9 V battery</td>
			<td style="width:227px;">Duracell</td>
			<td style="width:307px;">-</td>
			<td>used to power the LED&#39;s</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:187px;">horizontal electrophoresis chamber</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1704486EDU</td>
			<td>used to make and run agarose gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">vertical electrophoresis kit</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1658002EDU</td>
			<td>used to run polyacrylamide gels in calibration curves</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">power supply</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1645050&#160;</td>
			<td>used to power the electrophoresis</td>
		</tr>
	</tbody>
</table>

dna staining method based on formazan precipitation induced by blue light exposure

DNA 얼룩 메서드는 생물 의학 연구에 매우 중요 하다. 우리 설계 색된 침전의 형성에 의해 육안으로 DNA 시각화를 허용 하는 간단한 방법. 아크릴 아 미드를 몸을 담글 하 여 작동 또는 agarose DNA SYBR 녹색 1 x (2.0 µ M에 해당)의 해결책에 있는 젤 나 (SG 나) 보라색을 생성 하는 0.20 m m 블루 니트로 tetrazolium formazan 햇빛에 드러낼 때의 침전 또는 특별히 라이트 블루. 또한, DNA 복구 테스트가 우리의 방법으로 얼룩진 agarose 젤에는 암 피 실린 내성 플라스 미드를 사용 하 여 수행 되었습니다. 식민지의 많은 수와 함께 전통적인 보다 우리의 방법으로 얻은 SG를 사용 하 여 얼룩이 나 자외선 조명. 설명된 방법, 특정, 및 비-독성 DNA 검색, 시각화는 transilluminator 없이 "육안" 생체의 수 이며 저렴 하 고 필드 사용에 대 한 적절 한입니다. 이러한 이유로, 우리의 새로운 DNA 방법 얼룩이 지는 연구와 산업에 잠재적인 이점이 있습니다.

자주색 formazan 침전 (질량 분석13확인) DNA는 나타납니다. 실험에서 DNA 사다리 보정 곡선 (그림 3)을 로드 했습니다. 아크릴 및 agarose 젤에 대 한 프로토콜 작동 하지만 낮은 강도 있으며 agarose에서 더 긴 시간이 걸립니다. 그러나, agarose 젤의 사용 수 상용 키트를 사용 하 여 샘플의 복구 하 고 추출을 방해 하지 않습니다. 블루 Led와 함께...

Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

이 방법을 사용 하면 선택적 얼룩이 지 고 젤에서 DNA의 정량화 SYBR 녹색에서 젤을 몸을 담글 하 여 난 / 니트로 블루 Tetrazolium 솔루션 및 다음 햇빛 또는 파란색 광원을 노출. 이 보이는 침전 생성 하 고 필드 사용에 대 한 이상적 거의 장비가 필요 합니다.

DNA 방법 Formazan 강 수 푸른 빛의 노출에 의해 유도에 따라 얼룩

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

dna-staining-method-based-on-formazan-precipitation-induced-blue

Research

JoVE Journal

Biochemistry

9.4K 조회수.  Universidad de Chile. 이 방법을 사용 하면 선택적 얼룩이 지 고 젤에서 DNA의 정량화 SYBR 녹색에서 젤을 몸을 담글 하 여 난 / 니트로 블루 Tetrazolium 솔루션 및 다음 햇빛 또는 파란색 광원을 노출. 이 보이는 침전 생성 하 고 필드 사용에 대 한 이상적 거의 장비가 필요 합니다.

비디오: DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

블루 기본 페이지와 단백질 상관 관계 프로파일 링에 의한 선호도 정제 단백질 복합체 해결

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

연구

생화학

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Smartphone을 이용한 DNA 단편 분리 실시간 추적

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

근 적외선 형광 레이블이 DNA 아크릴 아 미드 젤 전기 이동 법으로 시각을 사용 하 여 DNA 중 합 효소 활동 분석 결과

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

꼬마 선 충 에서 높은 처리량 화학 심사 하는 간단한 방법

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

전기 화학 탐지의 중수소 활동적인 동위 원소 효과 Shewanella oneidensis 미스터-1에서 세포 외 전자 전송에

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

블루 기본 젤 전기 이동 법에 의해 Thylakoid 막 단백질 복합물의 분석

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

분광학 그리고 스캐닝 전자 현미경 검사 법에 의해 섬유 소 혈전 구조에 β 아 밀 로이드 유도 비정상의 분석

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

고처리량 프리마제 프로파일링을 사용하여 DNA 프리마제에 의한 DNA 서열 인식

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

격자 광 시트 현미경을 사용하여 표면 T 세포 수용체 역학 4 차원 시각화

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

네이티브 오토플루임션을 기반으로 sFLIM에 의한 식물 섹션의 형광 프로브와 리그닌 간의 상호 작용 측정

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...