우리는이 프로토콜을 개발과 지원 사이먼 Alberti 감사합니다. 이 작품은 국립 보건원 (GM25874), 하워드 휴즈 의학 연구소 Investigatorship (SL)을, 그리고 국립 과학 재단 predoctoral 훈련 부여 (RH)을에서 교부금에 의해 지원되었다.

<html> 1 부 : 겔 준비 <ol><li> 겔 주조 트레이를 조립. 수평 DNA 전기 영동 표준 장비를 사용할 수 있습니다. 샘플 많은 경우, 우리는 최대 4 개의 50 - 잘 빗과 함께 X 24cm 슬래브 20cm를 준비합니다. 이들은 얼룩 이미지를 왜곡 수 슬래브가 흠집이없는 있는지 확인하십시오. </li><li> 1X 태의 1.5 % 아가로 오스 솔루션 (중간 또는 높은 겔 강도, 낮은 EEO)를 만듭니다. 볼륨 완전히 잠수함 빗 치아를 충분히해야합니다 - 당신은 검색을 최대화하기 위해 가능한 한 많은 예제를로드하는 것이 좋습니다. 마이크로 웨이브는 혼합물은 아가로 오스는 완전히 녹아 때까지. </li><li> 신속하게 10 %의 주식의 0.1 %에 SDS를 추가합니다. 섞어 소용돌이. 일부 아가로 오스는이 단계 동안 굳은 경우, 뜨거운 접시를 사용하여 redissolve하고 끓는 않도록주의하십시오. </li><li> 캐스팅 트레이에 솔루션을 하거라. 그들이 나중에 전송을 방해할 수있는 것처럼 언제 거품이 밖으로 경사각에 빗을 사용합니다. </li><li> 젤 설정한 후, 빗을 제거하고 겔 탱크에 젤 넣습니다. 완전 잠수함 1X 태에서 젤은 SDS 0.1 %를 포함. </li></ol> 2 부 : 준비 샘플 <ol><li> 효모 lysates의 높은 처리량 분석을 위해, 우리는 96 블록에 잘 급속한 동요와 하룻밤 성장 2 - ML 문화로 시작합니다. 이 경우, 각 문화는 overexpressing 관심의 단백질이다. 낮은 풍부한 단백질을 분석하면, 더 큰 문화 볼륨을 사용해야합니다 </li><li> 상온에서 5 분 2000 RCF에서 원심 분리하여 세포를 수확. </li><li> 다시 물, 원심 분리기에서 세포를 Resuspend. </li><li> 1 ML spheroplasting 솔루션 Resuspend. 30 약 30 분 품어 ° C (당신은 현미경으로 spheroplasting 효율성을 확인 수 있습니다.) </li><li> 상온에서 5 분 800 RCF에서 원심 분리기. 완전히 뜨는 제거합니다. </li><li> 100 ML의 용해 버퍼 pelleted spheroplasts을 Resuspend. </li><li> 테이프 2 분 고속에 소용돌이와 함께 블록을 커버. </li><li> 2 분 4,000 RCF에서 펠렛 셀룰러 잔해. </li><li> 조심스럽게 새로운 용기, 예를 들어, 96 - 웰 플레이트 PCR에 뜨는 제거합니다. </li><li> 원하는 경우, lysates의 단백질 농도를 결정한다. </li><li> 1X 샘플 버퍼를 포함하는 lysates를 생성하는 샘플에 4X 샘플 버퍼를 추가합니다. 상온에서 5 분 알을 품다. </li><li> 젤을로드합니다. 원하는 경우, 샘플 볼륨의 절반을 저장하고 SDS - PAGE 분석을 위해 끓여. 나중에 양도의 범위를 모니터하기 위해, 사전에 묻은 SDS - PAGE 마커를 포함합니다. 또한, 매우 큰 단백질 (예, 닭고기 pectoralis 추출)로 구성된 분자량 마커가 해결 단지의 크기를 추정하는 데 사용할 수 있습니다. </li><li> 염료 앞에는 젤의 끝에서 ~ 1cm에 도달할 때까지 저전압 (≤ 3 V / cm 겔 길이)에서 실행합니다. 이것은 몇 시간이 소요됩니다. 젤 시원한 유지하는 것이 중요합니다, 그렇지 않으면 낮은 분자량의 단백질 (예 : 단량체)의 확산들은 검색을 제한할 수 있습니다. </li></ol> 파트 3 : 전송 <ol><li> 젤 같은 크기로 nitrocellulose의 한 조각을 잘라 버릴거야. </li><li> 젤 같은 크기로 GB004 20 조각과 GB002 압지 8 조각을 잘라 버릴거야. 심지로 사용될 GB004의 추가 부분을 잘라, 겔보다 약 20 cm 폭을합니다. </li><li> 1X TBS에서 nitrocellulose, 심지, 그리고 GB002 4 조​​각을 담가. </li><li> 마른 GB004 20 조각, 다음 마른 GB002 4 조​​각, 다음 사전 서부 유럽 표준시 GB002 중 하나 조각 : 플라스틱 용기에, 다음과 같이 서류의 스택을 조립. 이 스택의 상단에있는 nitrocellulose 있다고 믿는다. </li><li> 초과 실행 버퍼를 제거하는 물에 캐스팅 트레이 간략에서 젤 린스. 그런 다음, 조심스럽게 스택에 트레이에서 젤 슬라이드 시작합니다. 트레이에서 젤 슬라이딩 있지만, 필요에 따라 TBS와 멤브레인을 줄여봐. 추가 버퍼 젤 아래에 갇혀되는 것을 거품을 방지하는 데 도움이됩니다. 전송 피펫이 목적을 위해 잘 작동합니다. </li><li> 젤이 스택으로 이동되었습니다 후, 거품에 대해 철저히 확인하십시오. 어떤이있는 경우, 젤의 가장자리를 채취해서 거품이 해낼 수있을 때까지 버퍼를 다시 적용합니다. </li><li> 젤 위에 나머지 세 미리 침수 GB002 조각을 넣어. 단단히 스택의 맨 위에 피펫 롤링하여 모든 레이어 간의 접촉을 철저히 보장합니다. </li><li> TBS 담긴 두개의 높은 쟁반과 플렝크 전송 스택을. 심지의 양쪽 끝에이 TBS에 빠져들 것입니다 이러한 스택에 걸쳐 사전 서부 유럽 표준시 심지를 드레이프. </li><li> 추가 무게를 (예, 물 500 ML 병) 베어링 추가 플라스틱 트레이로 조립 전송 스택을 커버. </li><li> 세 시간이나 밤새 최소 진행 전송을 허용합니다. </li><li> 전송 후, 멤브레인 표준 양식은 모래 바닥으로 처리할 수 있습니다. </li></ol> Spheroplasting 솔루션 1.2 M D - sorbitol 0.5 MM MgCl 2 20 MM 트리스, 산도 7.5 50 MM의 BME (신선 추가) 0.5 MG / ML Zymolyase 100T는 (신선한 추가) coration : 밑줄; &quot;&gt; 용해 완충액 100 MM 트리스 7.5 50 MM NaCl 10 MM BME는 (신선한 추가) 단백 분해 효소 억제제는 (신선한 추가) 4X 샘플 버퍼 2X 태 20 % 글리세롤 8퍼센트 SDS 취향에 bromophenol 블루

<ol>
	<li>Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+[PSI+]+prion+aggregates+are+formed+by+small+Sup35+polymers+fragmented+by+Hsp104.">Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104.</a> J. Biol. Chem. 278, 49636-49643 (2003).</li><li>Alexandrov, I. M., Vishnevskaya, A. B., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+and+Propagation+of+Polyglutamine-based+Amyloids+in+Yeast:+TYROSINE+RESIDUES+ENABLE+POLYMER+FRAGMENTATION.">Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION.</a> J. Biol. Chem. 283, 15185-15192 (2008).</li><li>Allen, K. 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=Hsp70+chaperones+as+modulators+of+prion+life+cycle:+novel+effects+of+Ssa+and+Ssb+on+the+Saccharomyces+cerevisiae+prion+[PSI+].">Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].</a> Genetics. 169, 1227-1242 (2005).</li><li>Aron, R., Higurashi, T., Sahi, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J-protein+co-chaperone+Sis1+required+for+generation+of+[RNQ+]+seeds+necessary+for+prion+propagation..">J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation..</a> The EMBO journal. 26, 3794-3803 (2007).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+amyloid+aggregates+using+agarose+gel+electrophoresis.">Analysis of amyloid aggregates using agarose gel electrophoresis.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Borchsenius, A. S., Muller, S., Newnam, G. P., Inge-Vechtomov, S. G., Chernoff, Y. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+variant+maintained+only+at+high+levels+of+the+Hsp104+disaggregase.">Prion variant maintained only at high levels of the Hsp104 disaggregase.</a> Current Genetics. 49, 21-29 (2006).</li><li>Salnikova, A. B., Kryndushkin, D. S., Smirnov, V. N., Kushnirov, V. V., Ter-Avanesyan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense+suppression+in+yeast+cells+overproducing+Sup35+(eRF3)+is+caused+by+its+non-heritable+amyloids.">Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids.</a> J. Biol. Chem. 280, 8808-8812 (2005).</li><li>Tank, E. M., Harris, D. A., Desai, A. A., True, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+repeat+expansion+results+in+increased+aggregation+and+reveals+phenotypic+variability.">Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.</a> Mol. Cell. Biol. 27, 5445-5455 (2007).</li><li>Douglas, P. 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=Chaperone-dependent+amyloid+assembly+protects+cells+from+prion+toxicity.">Chaperone-dependent amyloid assembly protects cells from prion toxicity.</a> Proc. Natl. Acad. Sci. USA. 105, 7206-7211 (2008).</li><li>Nagy, B., Costello, R., Csako, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downward+blotting+of+proteins+in+a+model+based+on+apolipoprotein(a)+phenotyping.">Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping.</a> Analytical Biochemistry. 231, 40-45 (1995).</li></ol>

SDD - AGE 먼저 Kryndushkin 외 의해보고되었다. 1, SDS 방지 단지 공부를 [PSI +] 효모의 프리온, 이후 프리온이 아닌 프리온 집계 2-9 모두를 공부하고 널리 사용 발견했습니다. 그러나, 아가로 오스 겔에서 막 다음 전기 영동으로 단백질의 양도 문제이며, 왜곡된 얼룩 이미지 5 발생할 수 있습니다. 또한, 가장 일반적으로 사용하는 잠수 electroblotting 기술은 젤의 크기에 대한 실질적인 제한을 도입하기 때문에 처리할 수 샘플의 수입니다. 우리는 아래로 모세 전송 10, 겔에서 nitrocellulose 막에 단백질을 전송 마른 모래 바닥 논문의 스택을 사용하는 간단한 절차를 채용하여 이러한 문제를 해결했습니다. 모세관 전송 왜곡을 방지하고 대형 젤 쉽게 처리할 수 있습니다. SDD - AGE를 사용하기 전에 고려해야 할 몇 가지가 있습니다. 원유 샘플 (예 : lysates), 특정 단백질의 면역이 필요합니다. SDD - 나이가 충분히 관심의 단백질 단지를 변성하지 감지하는 단백질 (들)은 amyloidogenic 지역 이외의 에피토프 태그를 부담합니다 그럼. 그들이 정상적인 SDS - PAGE를위한 것입니다 같은 Lysates는 일반적으로 두 가지 중요한 차이점으로 준비하실 수 있습니다. 첫째, proteolysis에 의해 저하를 방지하기 위해 증가주의 이동합니다. 여기에 사용되는 부분 denaturing 조건은 프로 테아제를 inactivate에 충분하지 않은, 또한 proteolysis에 타겟 단백질이 더 취약 할 수 있습니다. 적어도 두 배 권장 농도에서 전체 테아제 억제제 칵테일을 사용합니다. 둘째, 샘플을 피해야한다 가열. 모든 단량체 음성 컨트롤이 필요한 경우, 예를 들어 높은 분자 - 중량 종은 monomeric 단백질 가장 amyloids를 복원하는 것입니다 공유 결합 수정, 95에서 10 분 배양 ° C를 사용할 수로 인해하지 않는지 확인합니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>zymolyase 100T</td>
<td>&nbsp;</td>
<td>Seikagaku America</td>
<td>120493-1</td>
<td>&nbsp;</td>
<tr>
<td>Halt Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>78429</td>
<td>&nbsp;</td>
<tr>
<td>Hybond-C extra nitrocellulose</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN303E</td>
<td>&nbsp;</td>
<tr>
<td>GB004 blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>10427926</td>
<td>&nbsp;</td>
<tr>
<td>GB002 (3MM Chr) blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-917</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (UltraPure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis

아밀로이드 집계는 conformationally 아르 다수의 단백질의 misfolding pathologies과 underlies prions의 전염성 속성 자체 템플릿 낮은 생물에 유익한 역할을 할 생각되고 있습니다 단백질을.과 관련된 Amyloids들은 불용성 및 구조 이질에 의한 연구에 악명 어려운되었습니다. 그러나, 크기 및 세제 불용성을 바탕으로 아밀로이드 고분자의 해상도에 의해 가능하게되었습니다 세미 Denaturing 세제 - 아가로 오스 겔 전기 영동을 (SDD - AGE). 이 기술은 아밀로이드 conformational 변종의 검출 및 특성에 대한 광범위한 사용을 찾는 것입니다. 여기, 우리는 소설 prions 및 기타 amyloidogenic 단백질에 대한 화면으로 대규모 애플 리케이션에서의 사용을 용이하게이 기술의 적응을 보여줍니다. 새로운 SDD - AGE 방법은 더 큰 안정성을 위해 모세 전송을 사용하고 사용의 용이성, 그리고 모든 크기의 겔이 수용 수 있습니다. 따라서, 세포 또는 정제된 단백질에서 준비한 샘플 많은가, 태그 단백질의 SDS - 불용성 conformers의 존재를 동시에 처리할 수 있습니다.

Watch this Scientific Journal Video about Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis at JoVE.com

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

SDD - 나이가 세포에서 고분자와 같은 아밀로이드의 감지 및 특성화를위한 유용한 기법입니다. 여기 우리는 대규모 응용 프로그램에이 기술을 의무하게 적응을 보여줍니다.

세미 Denaturing 세제 - 아가로 오스 젤 전기 영동에 의해 아밀로이드 집계에 대한 심사

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

Research

JoVE Journal

Biology

26.7K 조회수.  Whitehead Institute for Biomedical Research. SDD - 나이가 세포에서 고분자와 같은 아밀로이드의 감지 및 특성화를위한 유용한 기법입니다. 여기 우리는 대규모 응용 프로그램에이 기술을 의무하게 적응을 보여줍니다.

비디오: Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Denaturing 그라디언트 젤 전기 영동 (DGGE)

연구

생물학

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Denaturing 요소의 Polyacrylamide 젤 전기 영동 (우레아 페이지)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

셀룰러 Lysates에서 Multiprotein의 단지의 분석을 위해 블루 기본 Polyacrylamide 젤 전기 영동 (BN - PAGE)

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

상업 카세트를 재사용하여 물을 붓는과 단백질 젤 러닝

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

의 DNA 조각의 분리를위한 아가로 오스 젤 전기 영동

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

점액 아가로 오스 겔 전기 영동 : 높은 분자량 당 단백질에 대한 웨스턴 블로 팅


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

꼬마 선 충 나이 연구 변화 내재 단백질 집계에 방법

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal 현미경 검사 법 밝혀 세포 표면 수용 체 이미지 상관 분광학을 통해 집계

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

경작 된 인간 세포 블루 네이티브 Polyacrylamide 젤 전기 이동 법 및 Immunoblotting 사용 하 여에서 미토 콘 드리 아 호흡 사슬 단지의 분석

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...