이 작품은 혁신에 대한 캐나다 재단 (CFI), 보건 연구 (CIHR)의 캐나다 연구소와 자연 과학 및 캐나다 공학 연구 협의회 (NSERC)에서 보조금에 의해 지원되었다.

1 부 : 인플루엔자의 파괴 Virion <ol><li> 베크맨 옵티마에서 사용할 TLA - 120.2 로터에 맞게 설계된 하나 베크맨 폴리 카보 네이트 원심 분리기 튜브 (11mm X 34mm)로 MNT 버퍼의 750 μl (20 MM MES, 150 MM NaCl, 30 MM 트리스, 산도 7.5)를 추가 최대 - E의 초원 심 분리기. </li><li> 그 튜브로, 인플루엔자 바이러스 (2 MG / ML H3N2 X - 31 A/AICHI/68 변형) 500 μl를 추가합니다. 몇 시간을 아래와 pipetting하여 MNT 버퍼와 바이러스를 섞는다. </li><li> TLA - 120.2 로터를 사용하여 베크맨 옵티마 최대 - E의 원심 분리기에서 109,000 XG, 4 ° C에서 10 분 원심 분리기. </li><li> 뜨는을 제거하고 500 μl 중단 버퍼의 펠릿 (100 MM KCl, 5 MM MgCl 2, 5 % (W / V) 글리세롤, 50 MM의 octylglucoside, 10 MG / ML lysolecithin, 1.5 MM dithiothreitol, 100 MM MES를 resuspend 산도 5.5). </li><li> 와동 적극적으로, 그리고 31 흔들 ° C를 eppendorf thermomixer에 20 분. </li></ol> 2 부 : 글리세롤 기울기 세디먼트 속도 원심 분리 <ol><li> 1 ML 70% (V / V) 글리세롤, 0.75 ML 50 % 글리세롤, 0.375 ML 40 % 글리세롤, 그리고 1.8 : 베크맨 ultraclear의 원심 분리기 튜브 (13mm X 51mm)를로 글리세롤의 다음과 같은 금액을 배치하여 글리세롤 기울기 준비 ML 33 % 글리세롤. 글리세롤 솔루션은 NM 버퍼 (150 MM NaCl, 50 MM MES, 산도 5.5)와 순수한 글리세롤을 혼합하여 만들어집니다. 또한 글리세롤과 버퍼를 모두 포함하는 균형 튜브를 준비합니다. </li><li> 소용돌이 다시 바이러스 샘플을 중단하고, 글리세롤 기울기에 그것을로드합니다. </li><li> 베크맨 MLS - 50 스윙 - 물통 회전자에 217,000 XG, 4 ° C에서 3.75 시간 동안 그라데이션을 원심 분리기. </li><li> 원심 분리가 완료되면 수동으로 튜브의 상단에서 시작 그라디언트 250 μl aliquots를 수집합니다. ° C. 얼음이나 4 분수 유지 </li></ol> 파트 3 : SDS의 Polyacrylamide 젤 전기 영동에 의해 글리세롤의 그라데이션 분수 분석 <ol><li> 각 분획에서 신선한 튜브 20 μl를 제거합니다. </li><li> 5 배 SDS - PAGE 샘플 버퍼의 5 μl를 추가합니다. </li><li> 95 열 ° C 5 분. </li><li> 간단히 샘플을 스핀 다운하고 10 % polyacrylamide 젤로 그들을로드하고, 우물 중 하나 분자량 마커를 포함합니다. </li><li> bromphenol 블루 로딩 염료가 젤 하단에 가까운 도달 때까지 겔에서 샘플을 실행합니다. </li><li> 쿠매시과 청색의 젤 얼룩. </li></ol> 파트 4 : vRNP 분수의 농도 <ol><li> 주로 NP가 들어있는 글리세롤의 분수를 선택, 그들을 결합, 두 베크맨 폴리 카보 네이트 원심 분리기 튜브 (11mm X 34mm)로 그들을 배포합니다. </li><li> 디에틸 pyrocarbonate (DEPC) 처리된 Ultrapure 물, 그리고 pipet 위아래 섞어 여러 번 각 튜브를 입력합니다. </li><li> 베크맨 TLA - 120.2 로터에 157,000 XG, 4 ° C에서 4.5 시간 동안 원심 분리기. </li><li> 뜨는을 제거하고, DEPC - 처리된 물을 50 μl의 펠렛을 resuspend. </li><li> 원하는 경우, 집중 vRNPs의 280도 측정할 수 있습니다. NP는 풀링된 분수의 주요 구성 요소 존재로서, NP의 molarity의 견적가 55,350 자사의 흡광 계수 M -1 cm -1 (같은 ProtParam 3를 통해 결정)를 사용하여 계산하실 수 있습니다. </li><li> 나누어지는 정화 vRNPs을하고, 그들 ° C 나중에 사용하기 위해 -80에서 냉동 보관합니다. </li></ol> 제 5 부 : vRNPs의 음성 스테 이닝 <ol><li> 9 μl로 정화 vRNP 1 μl를 희석 갓 만든 여과 - 두 번 MNT 버퍼 (20 MM MES, 150 MM NaCl, 30 MM 트리스, 산도 7.5). 다른 버퍼는 또한 vRNPs을 희석하고 표본 그리드 (단계 5.5) 씻어 확인을하지만, uranyl 아세테이트가 부정적인 얼룩에 사용하는 경우 PBS를 사용하지 마십시오. </li><li> 30 초 동안 parlodion 탄소 필름으로 이전 코팅 글로우 방전 표본 (구리 TEM)은 그리드. </li><li> 핀셋으로 신선한 형광 방전 표본 격자를 보유하고 표본 격자에 희석 vRNP의 5 μl 드롭을 적용합니다. </li><li> 팔분을위한 그리드에 시편의 드롭를 남겨주세요. </li><li> 기다리는 동안 벤치에 Parafilm의 작은 스트립을 배치, 10 μl MNT 버퍼의 각 (격자를 씻어), 그리고 갓 만든 얼룩 솔루션의 80 μl 드롭 (1 % 암모늄 molybdate 또는 1을 포함하는 두 방울을 분배 Parafilm에 %의 uranyl 아세테이트). </li><li> 흡착의 8 분 후에, 필터 종이로 표본 그리드 (삼각형으로 잘라)에서 샘플 솔루션의 일부를 심지. 표본의 격자가 건조시키지 마십시오. </li><li> 일분의 총 시간을위한 10 μl MNT 버퍼를 각각 포함하는 두 방울의 표본 격자를 씻으십시오. 이것은 신중하게 드롭에 표본 그리드를 낮추고, 그리고 필터 종이로 표본 그리드 (표본 그리드 건조 아무말도없이)에서 샘플 솔루션의 일부를 위킹 통해 이루어집니다. </li><li> 즉시 표본 격자에서 버퍼의 마지막 한 방울을 위킹 후, 완료정의 잠수함은 표본의 얼룩 비말 내에서 격자 1 잠깐 만요. </li><li> 완전 필터 종이로 표본 그리드에서 얼룩 솔루션을 심지. </li><li> 전송 전자 현미경으로 관찰하기 전에 몇 분 동안 공기 건조 표본의 격자를 허용합니다. </li></ol> 6 부 : 대표 결과 : <img src="/files/ftp_upload/old/1105_2009_1_22_1/1105_Figure2.png" alt="그림 2" width="600" height="215" /> 그림 1 독감 NP (~ 56 KDA)와 같은 바이러스성 ribonucleoprotein의 단지에있는 주요 단백질은, NP 밴드는 일반적으로 vRNPs (그림 1)이있는 분수에서 가장 강한 밴드이다. NP 이외에도, 각 인플루엔자 vRNP 또한 trimeric RNA 효소 (82, 86 및 86.5 KDA의 분자 가중치) 복합의 사본을 포함하고 있습니다. 이것은 수 또는 풍부가 낮은 NP의 비교이기 때문에 쿠매시 푸른 얼룩으로 표시되지 않을 수 있습니다. polymerases 그러나, 대신에 얼룩 Commassie 파란색의 경우, 은색이 젤이 수행되는 중 얼룩 검색하실 수 있습니다. 독감 매트릭스 단백질 M1은 (~ 28 KDA) NP 단백질 피크 분수가 존재하는 분수에 최소한 존재해야합니다. <img src="/files/ftp_upload/old/1105_2009_1_22_2/1105_3.png" alt="그림 3" width="600" height="360" /> 그림 2 전송 전자 현미경 하에서 시각으로 부정 묻은 vRNPs 길이가 120 나노미터 (그림 2A)에 약 30 nm의 아르 가변 길이 막대 모양의 입자 유사 vRNPs를 얻을 것입니다. oligomeric NP가 더욱 더블 헬리컬 반복 구조로 접혀 있습니다 NP 분자의 체인으로 구성되어 있습니다, 이러한 막대 모양의 입자 중 하나를 끝에 고리 때로는 (그림 2B) 볼 수 있습니다.

<ol>
	<li>Kemler, I., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+microinjected+influenza+virus+ribonucleoproteins.">Nuclear import of microinjected influenza virus ribonucleoproteins.</a> Virology. 202, 1028-1033 (1994).</li><li>Wu, W. W. H., Weaver, L. L., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+the+nuclear+localization+sequences+on+influenza+a+ribonucleoprotein+complexes.">Ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes.</a> J Mol Biol. 374, 910-916 (2007).</li><li>Gasteiger, E., 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> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Wu, W. W. H., Sun, Y. H. B., Pant&#233;, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+influenza+A+viral+ribonucleoprotein+complexes+is+mediated+by+two+nuclear+localization+sequences+on+viral+nucleoprotein.">Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein.</a> Virol J. 4, 49-49 (2007).</li><li>Babcock, H. P., Chen, C., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-particle+tracking+to+study+nuclear+trafficking+of+viral+genes.">Using single-particle tracking to study nuclear trafficking of viral genes.</a> Biophys J. 87, 2749-2758 (2004).</li></ol>

vRNPs의 정화가 Kemler 외. (1994)에 기술된 절차에 따라 달라집니다. 한 우리는 다른 사람들이 그들이 핵 수입을 공부 vRNPs을 분리하기 위해이 프로토콜을 사용하고 있습니다. 2,4,5 우리는 바이러스 게놈은 RNA로 구성되어 있기 때문에 RNase 무료 도움말 및 vRNPs을 조작 튜브의 사용을 권장하고, 따라서 RNA의 존재에서 쉽게 떨어집니다. 또한, 모든 버퍼는 RNase ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>H3N2 X-31 A/AICHI/68 influenza A</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>490715</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M-8250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G33-1</td>
<td>&nbsp;</td>
<tr>
<td>Octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O-8001</td>
<td>&nbsp;</td>
<tr>
<td>Lysolecithin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4129</td>
<td>&nbsp;</td>
<tr>
<td>Dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-9779</td>
<td>&nbsp;</td>
<tr>
<td>Coomassie brilliant blue G-250</td>
<td>&nbsp;</td>
<td>Kodak</td>
<td>1367796</td>
<td>&nbsp;</td>
<tr>
<td>diethyl pyrocarbonate (DEPC)-treated water</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>750024</td>
<td>&nbsp;</td>
<tr>
<td>Uranyl Acetate</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>19481</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Molybdate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A-674</td>
<td>&nbsp;</td>
<tr>
<td>Optima MAX-E Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>434491</td>
<td>&nbsp;</td>
<tr>
<td>MLS-50 Rotor Package, Swinging Bucket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>367280</td>
<td>&nbsp;</td>
<tr>
<td>TLA-120.2 Rotor Assembly, Fixed-Angle, Titanium</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362046</td>
<td>&nbsp;</td>
<tr>
<td>Eppendorf Thermomixer</td>
<td>&nbsp;</td>
<td>Brinkman</td>
<td>022670000</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

purification and visualization of influenza a viral ribonucleoprotein complexes

인플루엔자 바이러스 게놈은 개별적으로 바이러스성 ribonulceoprotein 입자 (vRNPs)에 인플루엔자 핵 단백질 (NP)의 복사본이 여러 개 모여 팔 부정적 - 감각, 단일 좌초 RNA 분자로 이루어져 있습니다. 인플루엔자 vRNPs는 바이러스 봉투 안에 동봉됩니다. 그들이 호스트 핵 수송 기계에 액세스할 어디 셀 항목 동안 그러나, 이러한 vRNP의 단지는 세포질에 발표됩니다. 인플루엔자 vRNPs 및 인플루엔자 게놈의 복제의 핵 수입을 연구하기 위해서는, 그것은 바이러스의 다른 구성 요소가 이러한 프로세스를 방해하지 않도록 격리 v​​RNPs 작동하는 데 유용합니다. 여기, 우리는 인플루엔자 바이러스로부터 vRNPs 정화 절차를 설명합니다. 절차는 싸여 virion에서 vRNP 단지를 공개하기 위해 인플루엔자 세제과 virion의 장애로 시작합니다. vRNPs은 다음 인플루엔자 속도 침강에 의해 33~70% 불연속 글리세롤 기울기에 virion의 다른 구성 요소에서 분리됩니다. 글리세롤 기울기에서 얻은 분수는 다음 쿠매시과 청색의 얼룩 후 SDS - PAGE를 통해에서 분석하고 있습니다. NP가 포함된 정상 분수 그런 다음 함께 풀링 및 원심 분리에 의해 집중되고있다. 농도 후 vRNPs의 무결성은 부정적인 얼룩 후 전송 전자 현미경에 의해 vRNPs의 시각화에 의해 확인됩니다. 글리세롤 기울기 정화는 Kemler에게서의 수정입니다<em&gt; 외.</em&gt; (1994)<sup&gt; 1</sup&gt;, 그리고 부정적인 얼룩이 우에 의해 수행되었습니다<em&gt; 외.</em&gt; (2007).<sup&gt; 2</sup

Watch this Scientific Journal Video about Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes at JoVE.com

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

인플루엔자 바이러스의 게놈은 RNA와 단백질 여덟 별도 단지, 칭했다 바이러스성 ribonucleoprotein 컴플렉스 (vRNPs)로 구성되어 있습니다. 본 논문은 글리세롤 기울기 정화 및 독감 vRNPs의 전송 전자 현미경의 시각화에 대해 설명합니다.

인플루엔자 바이러스성 Ribonucleoprotein의 단지의 정화 및 시각화

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

purification-visualization-influenza-viral-ribonucleoprotein

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12.9K 조회수.  University of British Columbia - UBC. 인플루엔자 바이러스의 게놈은 RNA와 단백질 여덟 별도 단지, 칭했다 바이러스성 ribonucleoprotein 컴플렉스 (vRNPs)로 구성되어 있습니다. 본 논문은 글리세롤 기울기 정화 및 독감 vRNPs의 전송 전자 현미경의 시각화에 대해 설명합니다.

비디오: Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

연구

생물학

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

자기 공명 영상 준비, 정화하고, 명암 요원 이용 란탄족 단지의 특성화

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104의 정화, 단백질 Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

높은 활성 효모 변이의 색층 정화

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

RIP 칩을 사용하여 세포 추출물에서 mRNAs, microRNAs 및 Ribonucleoprotein 단지의 단백질 구성 요소의 분리 및 확인에 방법

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

셀 벽의 탄성 특성의 AFM 기반의 매핑 : 조직, 세포 및 세포 이하의 해상도에서

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

조직 해리 차동 여과 이식 미토콘드리아의 신속한 분리 및 정제

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

세대와 인간의 INO80 염색질 리모델링 단지 및 서브 콤플렉스의 정화

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

마우스 모델의 유효성 검사는 세포 고유의 방식으로 LINC 단지를 방해

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...