이 프로젝트는 국립 과학 재단 (National Science Foundation) 및 Biomolecular 과학 협회에서 CJJ 부여에 의해 지원되었다.

1. 1 일 : 녹농균의 MexB는 pFB101에 의해 인코딩됩니다. MexB 유전자가 증폭되었다 P.부터 녹농균이라는 박테리아의 게놈의 DNA와 NdeI과 pET30b + 벡터의 XhoI 제한 사이트에 삽입. 구조는 C - 터미널 hexahistidine 태그를 포함하고 있습니다. <ol><li> 플라스미드가 E.을 변환하는 데 사용됩니다 대장균은 C43 (DE3) 12 변형율과 transformants 30 UG / ML kanamycin이 포함된 LB 한천에 도금입니다. </li></ol> 2. 일 2 : 숙박 문화 : <ol start="2"><li> 저녁에는, 30 UG / ML kanamycin이 포함된 4 X 3 ML의 LB 문화는 신선한 transformant의 식민지에서 주사를하고 있습니다. 또는 문화가 얼어 파마에서 주사 수 있습니다. 이러한 작은 문화가 ° C 야간 37 롤러에 성장하고 있습니다. </li></ol> 3. 3 일 : 6 리터 문화를 성장 : <ol start="3"><li> 아침에, 30 UG / ML kanamycin을 포함한 150 ML의 LB를 예방하기 위해 야간 문화를 사용합니다. 37 흔드는에서 ° C에서 문화를 성장. </li><li> 오후에는, 30 UG / Fernbach의 flasks의 ML의 kanamycin이 포함된 6 X 1L 2XYT 미디어를 예방하기 위해 작은 문화를 사용합니다. (1시 40분 희석을위한 문화 당 25 ML을 사용합니다.) 37 문화를 성장 ° C들은 1.5 시간 약 0.4-0.6의 OD 600에 도달할 때까지 </li><li> 문화가 적절한 밀도에 도달하면, 0.5 ML 1M IPTG를 추가하여 단​​백질 발현을 유도. 쉐이크에 다시 모든 flasks을 넣고 ° C 야간 30 그들을 계속 성장하고 있습니다. </li></ol> 4. 일 4 : 전지를 착취하고 단백질을 퓨리화잉 : <ol start="6"><li> 다음과 같이 버퍼 솔루션 테아제 억제제, DNAse하고, 라이 소 자임 추가 : 셀 resuspension 버퍼 50 ML하기 위해, 10 MG DNaseI (0.1 MG / ML 최종 농도), 라이 소 자임 1 완전한 EDTA (에틸렌 다이아 민 테트라 초산) 무료 테아제 억제제의 정제 및 핀치를 추가 . 막 resuspension 버퍼 60 ML 위해 1 테아제 억제제의 정제를 추가합니다. 막 resuspension 버퍼의 또 다른 50 ML하려면 1 테아제 억제제의 정제를 추가합니다. 얼음 세 솔루션을 유지. </li><li> 문화에게 세포를 수확하는 대규모 원심 분리기에서 30 분 5,000 RPM을 원심 분리기. </li><li> 100 ML 세포 resuspension 버퍼 (라이 소 자임 50 MM 낮잠, 산도 7.0, 300 MM NaCl, 2 MM MgCl 2, 1 완료 EDTA (에틸렌 다이아 민 테트라 초산) 무료 테아제 억제제의 정제, 0.1 MG / ML DNAse I, 핀치)에 세포를 Resuspend </li><li> 12,000 PSI (762 게이지 압력)에 프랑스의 압력 셀을 통해 두 번 세포 솔루션을 전달합니다. 병에서 세포 lysate를 수집 얼음이 차가운 유지. </li><li> SS34 원심 분리기 튜브에 세포 lysate를 전송하고 4 10,000 RPM에서 30 분 세포 파편을 제거하기 위해 원심 분리기 ° C를 SS - 34 로터 인치 </li><li> 조심스럽게 Ti647.5 초원 심 분리기 튜브에 뜨는를 제거합니다. 4C에서 40,000 rpm으로 50 분 원심 분리기. 뜨는 폐기하십시오. </li><li> 약에서 세포 세포막이 들어있는 펠렛을 Resuspend. 막 resuspension 버퍼의 25 ML (50 MM 낮잠, 산도 7.0, 300 MM NaCl, 5 % 글리세롤, 1 완료 EDTA (에틸렌 다이아 민 테트라 초산) 무료 테아제 억제제의 정제). </li><li> 4시 50 분 Ti647.5 로터에서 40,000 rpm으로 깨끗한 원심 분리기 튜브와 원심 분리기에 막 현탁액을 전송 ° C. </li><li> 뜨는을 취소하고 25 ML 막 resuspension 버퍼에 씻어 멤브레인 펠릿 (50 MM 낮잠, 산도 7.0, 300 MM NaCl, 5 % 글리세롤, 1 완료 EDTA (에틸렌 다이아 민 테트라 초산) 무료 테아제 억제제 정제) resuspend. </li></ol> 5. TM 단백질 Solublization : <ol start="15"><li> resuspended 점막 (25 ML에 대한)에 6 ML 10 % DDM (최종 세제 농도 = 2 %의 DDM) 록 4 혼합물 ° C 2 시간.을 추가 </li><li> 4 40 분 40,000 RPM의 혼합물을 원심 ° C 불용성 단백질의 수용성 단백질 세제 단지를 분리 Ti647.5 로터 인치 MexB 단백질 세제 단지를 포함 뜨는을 저장합니다. </li></ol> 6. 아이맥 : <ol start="17"><li> resuspension 버퍼에 equilibrated 탈론 금속 친화 비즈와 고속 회전에서 얻은 뜨는을 섞는다. 4 롤러에 1 시간을위한 품어 ° C. </li><li> 중력 흐름 열 본문에 슬러리를 붓고과를 통해 흐름을 폐기. </li><li> 아이맥 바인딩 및 씻으 버퍼 20 ML (10 열 볼륨) (50 MM 낮잠, 산도 7, 300 MM NaCl, 5 % 글리세롤, 0.2 % DDM)와 컬럼을 씻어 </li><li> 아이맥 용출 버퍼 (50 MM 낮잠, 산도 7.0, 300 MM NaCl, 5 % 글리세롤, 250 MM 이미다졸, 0.2 % DDM)와 단백질 Elute. </li><li> 용출 분수 15 μL 샘플을 가지고, polyacrylamide 젤 전기 영동에 의해 분석 15 μL 2X SDS 샘플 버퍼로 각각 섞는다. microcentrifuge에 30sec를 스핀. 각 분획의 MexB의 금액과 순결을 추정하기 위해 10 % polyacrylamide SDS 겔에서 샘플을 분석할 수 있습니다. </li><li> MexB 단백질 세제 단지와 4 회전 농축기에서 그들을 집중을 포함하고있는 분수 수영장 ° C.는 단백질이의 시에 침전하지 않는주의tep. </li></ol> 7. 겔 여과 칼럼 : <ol start="23"><li> 24 ML 실행 버퍼 (50 MM 낮잠, 산도 7.0, 300 MM NaCl, 5 % 글리세롤, 0.2 % 베타 octylglucoside)와 Superose 12 HL 10분의 30 컬럼을 사전 평형 및 평면 기준 나올 때까지 기다리는 것이 좋습니다. </li><li> 실행 버퍼 악타 시스템 로딩 루프 린스. </li><li> 그 결과 해당 컬럼에 그것을 적용하기 전에 주사기 필터를 사용하여 단백질 솔루션을 필터링합니다. </li><li> 5mg / ML 최대의 단백질 농도와 열의에 단백질 용액 240 μL까지로드합니다. </li><li> 버퍼의 1.5 열 볼륨 (36 ML)를 실행, 0.25 ML 분수를 수집합니다. 용출 량의 15 ML - MexB 단백질 세제 단지는 10 시경 최고로 elute해야합니다. </li><li> 정상 분수 5 μL 샘플을 가져가라. 2X SDS 샘플 버퍼 각 샘플 1시 1분를 섞습니다. 각 분획의 MexB의 금액과 순결을 추정하는 10 % polyacrylamide 젤의 샘플을 분석 </li><li> 순수 MexB를 포함하는 분수 수영장. </li></ol> 8. 대표 결과 : 그림 1은 겔 여과 칼럼에서 아이맥 열 및 개별 분수에서 풀링된 열 분수와 polyacrylamide 젤이 포함되어 있습니다. 겔 여과 칼럼 후 단백질 쿠매시 스테인드 polyacrylamide 젤로 순수한 나타납니다. 그림 2는 칼럼에서 단백질 세제 콤플렉스 eluting의 주봉을 보여주는 겔 여과 칼럼에서 추적을 포함하고 있습니다. MexB 단백질의 평균 수확량은 2XYT 문화의 6리터 당 약 2 MG입니다. <img src="/files/ftp_upload/2134/2134fig1.jpg" alt="그림 1" /> 그림 1. SDS - PAGE MexB의 PDCs의 정화의 젤. 레인 1, 분자량 마커. 2, 아이맥 분수를 풀링. 3-7, 젤 여과 칼럼 분수. <img src="/files/ftp_upload/2134/2134fig2.jpg" alt="그림 2" /> 그림 2. MexB 단백질 세제 단지 (PDC)에 대해 젤 여과 결과의 예.

<ol>
	<li>Eda, S., Maseda, H., Nakae, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+elegant+means+of+self-protection+in+gram-negative+bacteria+by+recognizing+and+extruding+xenobiotics+from+the+periplasmic+space.">An elegant means of self-protection in gram-negative bacteria by recognizing and extruding xenobiotics from the periplasmic space.</a> J. Biol. Chem. 278, 2085-2088 (2003).</li><li>Li, X. Z., Ma, D., Livermore, D. M., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+efflux+pump(s)+in+intrinsic+resistance+of+Pseudomonas+aeruginosa:+active+efflux+as+a+contributing+factor+to+beta-lactam+resistance.">Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to beta-lactam resistance.</a> Antimicrob. 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Bacteriol. 178, 306-308 (1996).</li><li>Srikumar, R., Kon, T., Gotoh, N., Poole, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Pseudomonas+aeruginosa+multidrug+efflux+pumps+MexA-MexB-OprM+and+MexC-MexD-OprJ+in+a+multidrug-sensitive+Escherichia+coli+strain.">Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain.</a> Antimicrob. Agents Chemother. 42, 65-71 (1998).</li><li>Tikhonova, E. B., Zgurskaya, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AcrA,+AcrB,+and+TolC+of+Escherichia+coli+Form+a+Stable+Intermembrane+Multidrug+Efflux.">AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux.</a> Complex. J. Biol. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-production+of+proteins+in+Escherichia+coli:+mutant+hosts+that+allow+synthesis+of+some+membrane+proteins+and+globular+proteins+at+high+levels.">Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.</a> J. Mol. Biol. 260, 289-298 (1996).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이온 전달, 세포 - 세포 통신, 소포 수송, 세포 구조의 유지 보수 및 호스트 병원체 상호 작용을 포함한 저항, 많은 중요한 세포 활동을 multidrug뿐만 아니라, 세포막에 포함된 단백질을 포함하고 있습니다. Transmembrane 단백질 알려진 단백질의 30 % 이상 만들어 사용 오늘날 의약품의 대다수에 대한 대상입니다. transmembrane 단백질의 접힘이나 부적 절한 활동은 낭성 섬유증과 당뇨병을 포함하여 중요한 ?...

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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>SDS sample buffer</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>161-0737</td>
<td>&nbsp;</td>
<tr>
<td>C43(DE3) E. coli strain</td>
<td>&nbsp;</td>
<td>Lucigen</td>
<td>60345-1</td>
<td>&nbsp;</td>
<tr>
<td>kanamycin sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>K4378</td>
<td>&nbsp;</td>
<tr>
<td>2XYT media</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP2466-2</td>
<td>&nbsp;</td>
<tr>
<td>LB media</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC61189-5000</td>
<td>&nbsp;</td>
<tr>
<td>IPTG</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I6758</td>
<td>&nbsp;</td>
<tr>
<td>DNaseI</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP3226-1</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L7651</td>
<td>&nbsp;</td>
<tr>
<td>Complete EDTA-free protease inhibitor tablets</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 873 580 001</td>
<td>&nbsp;</td>
<tr>
<td>NaP monobasic</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S6566</td>
<td>&nbsp;</td>
<tr>
<td>NaP dibasic</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5136</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M1028</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-1</td>
<td>&nbsp;</td>
<tr>
<td>n-dodecyl-&beta;-D-maltopyranoside</td>
<td>&nbsp;</td>
<td>Anatrace</td>
<td>D310</td>
<td>&nbsp;</td>
<tr>
<td>15ml tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>&nbsp;</td>
<tr>
<td>See-Saw Rocker</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SSL 4</td>
<td>&nbsp;</td>
<tr>
<td>Talon metal affinity resin</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>635503</td>
<td>&nbsp;</td>
<tr>
<td>imidazole</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I5513</td>
<td>&nbsp;</td>
<tr>
<td>10% polyacrylamide SDS PAGE gels</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-1454</td>
<td>&nbsp;</td>
<tr>
<td>Tris/glycine/SDS PAGE running buffer</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0732</td>
<td>&nbsp;</td>
<tr>
<td>Kaleidascope prestained molecular weight markers</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0324</td>
<td>&nbsp;</td>
<tr>
<td>Superose 12 30/10 column</td>
<td>&nbsp;</td>
<td>GEHealthcareSuperose</td>
<td>12 10/300 GL</td>
<td>&nbsp;</td>
<tr>
<td>Amicon centrifugal concentrator</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC801024</td>
<td>&nbsp;</td>
<tr>
<td>Syringe filter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SLFG R04 NL</td>
<td>&nbsp;</td>
<tr>
<td>Fernbach flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-552-39</td>
<td>&nbsp;</td>
<tr>
<td>Shaker to hold Fernbach flasks</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Akta system</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>J6 Large scale centrifuge with JLA-8.1000 rotor</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 l centrifuge bottles</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>969329</td>
<td>&nbsp;</td>
<tr>
<td>RC-5 centrifuge</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SS34 fixed-angle rotor and tubes</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sorvall floor model Ultracentrifuge</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T647.5 rotor and tubes with caps</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>08322</td>
<td>&nbsp;</td>
<tr>
<td>French Pressure Cell</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>FA-032</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

expression, detergent solubilization, and purification of a membrane transporter, the mexb multidrug resistance protein

Multidrug 저항 (MDR), 구조적 및 기능적으로 관련이없는 안티 - 암 약물이나 항생제의 다양한 내성이 될 수있는 암 세포 또는 병원체의 능력이, 공중 보건의 현재의 심각한 문제입니다. 이 multidrug 저항은 에너지 의존 약물 유출 펌프에 크게 때문입니다. 펌프는 독성이 임계값 아래에 자신의 세포 농도를 낮추고, 외부 매체에 방지 암 약물이나 항생제를 추방. 우리는 예를 들어, 화상 또는 낭성 섬유증, 또한 단백질 손상 암, 투석과 이식 환자에서 녹농균, 부상 또는 질병의 많은 종류의 환자에서 감염을 일으키는 기회 세균 병원체에 multidrug 저항을 연구하고있다. P.의 주요 MDR의 유출 펌프 녹농균이라는 박테리아는 내부 멤브레인 프로톤 - 약물 antiporter (RND), 외부 멤브레인 채널 (OMF) 및 periplasmic 링커 단백질 (MFP) 1-8 구성되어 삼자 간의 단지입니다. RND 및 OMF 단백질 transmembrane 단백질입니다. Transmembrane 단백질은 모든 단백질의 30 % 이상을 구성하고 현재의 약물 타겟의 65 %입니다. 소수성 transmembrane 도메인은 수성 버퍼에 단백질이 불용성합니다. transmembrane 단백질을 정화하기 전에, 그것은 단백질이 단백질 세제 단지 (PDC) 9-11로 solubilized 수 있도록 가벼운 세제를 포함하는 버퍼 조건을 찾을 필요가 있습니다. 이 예제에서, 우리는 RND 단백질, P.을 사용 , transmembrane 단백질의 재조합 형태로 표현이 세제를 사용 solubilize 다음 단백질 세제 단지를 정화하는 방법을 보여주는 녹농균이라는 박테리아의 MexB의 transmembrane 수송. 이것은 일반적인 방법은 표현, 정화 및 기타 여러 recombinantly 표현 막 단백질의 solubilization 적용할 수 있습니다. 단백질 세제 단지는 나중에 X - 선 결정 구조의 결정이나 crosslinking 연구를 포함한 생화 학적 또는 biophysical 특성화 사용할 수 있습니다.

Watch this Scientific Journal Video about Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein at JoVE.com

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

이 프로토콜에서 우리는 가용성 단백질 세제 복합으로 표현, solubilization, 그리고 recombinantly 표현 막 단백질, MexB의 정화를 보여줍니다. MexB는 기회 세균성 병원체 녹농균에서 multidrug 저항 막 수송이다.

표현, 세제 Solubilization 및 멤브레인 전송, MexB의 Multidrug 저항 단백질의 정제

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

expression-detergent-solubilization-purification-membrane-transporter

Research

JoVE Journal

Biology

21.2K 조회수.  University of Illinois Chicago - UIC. 이 프로토콜에서 우리는 가용성 단백질 세제 복합으로 표현, solubilization, 그리고 recombinantly 표현 막 단백질, MexB의 정화를 보여줍니다. MexB는 기회 세균성 병원체 녹농균에서 multidrug 저항 막 수송이다.

비디오: Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections.  Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains.  The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB.  The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB.  In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

MODS의 결핵의 진단 방법과 내성 결핵을 multidrug

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

연구

생물학

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

의 정화 M. magneticum 스트레인 AMB - 1 Magnetosome 관련 단백질 MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

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 ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

의 낭성 섬유증의 Transmembrane의 전도성 조절 단백질의 표현과 정제 Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

형광 단백질 분해 효소 보호를 사용하여 막 단백질 토폴로지를 결정 (FP​​P)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...