우리는 기꺼이 시니 Miettinen, 미나 Pöllänen 및 한인 Rautavesi의 기술 지원을 인정합니다. 우리는 HEK에게 녹음 장비를 제공하는 트윈 연쇄상 구균 - 태그 GFP 및 페카 Evijärvi을 표현하는 293 세포를 제공 헬카 Nurkkala 감사합니다. 이 작품은 핀란드의 아카데미에 의해 투자되었다, 번호 138329, 134684 및 258978을 부여합니다.

1. 비스와 패 혈성-Tactin 폴리 메타 크릴 수지 (술포 숙신 이미 딜)의 가교 수베 (BS3) <ol><li> 실온 BS3 가교제의 2 ㎎을 함유 한 밀봉 된 마이크로 튜브를 평형화. 주의 : BS3는 유해 물질이다. 보호 장갑과 고글을 착용 할 것. </li><li> 재현 탁 패 혈성-Tactin의 폴리 메타 크릴 레이트 수지 (100 MM 트리스 - 염산, 산도 8.0의 50 % 현탁액, 1 mM의 EDTA, 150 mM의 염화나트륨)에 의한 간단한 격렬한 흔들림 즉시에 피펫 팁을 사용하여 스핀 컬럼으로 서스펜션의 600 μl를 전송 끝은 잘라. </li><li> 실온에서 30 초 동안 1,500 XG에 원심 분리기. 흐름을 통해 폐기하고 열을 인산염 완충 생리 식염수 (PBS)의 450 μl를 추가합니다. </li><li> 완전히 PBS로 트리스 버퍼를 교체하고 마지막으로 원심 분리 한 후 pH를 8.0로 조정 PBS 430 μL에 수지를 떠나 이전 단계 2를 번 더 반복합니다. </li><li> (100)를 포함하는 피펫 팁 BS3으로 모세관의 호일에 구멍을1, 초순수의 L. 부드럽게 아래로 피펫에 의해 물에 BS3 분말을 녹여 바로 스핀 열 용액 20 μl를 추가합니다. 가교 반응에 BS3의 최종 농도는 ~ 1.2 ㎜이다. </li><li> 실온에서 30 분 동안 열을 돌립니다. 수지가 BS3 솔루션을 적절하게 혼합되어 있는지 확인합니다. </li><li> 반응을 해소하기 위해, 3M 트리스 - 염산, 산도 7.5의 10 μl를 추가하고 실온에서 또 다른 15 분 동안 열을 돌립니다. </li><li> 실온에서 30 초 동안 1,500 XG에 원심 분리기. 흐름을 통해 폐기 및 트리스 450 μL의 가교 수지를 재현 탁은 트윈 20 (TBST)와 완충 식염수. 원심 분리를 반복하고 단계를 두 번 더 씻는다. 마지막 단계에서, TBS 450 μL에 재현 탁 수지. </li><li> 끝으로 피펫 팁을 사용하여 새로운 튜브에 수지 현탁액을 전송 차단. TBS의 또 다른 450 μL와 컬럼에 남아있는 수지를 재현 탁하고 같은 튜브에 전송할 수 있습니다. 반복 일한 번 더 튜브에 열에서 수지의 최대 전송을 보장하기 위해 전자 마지막 단계. </li><li> 관을 실온에서 10 분간 방치하고 과량의 TBS를 제거하여 600 μL로 볼륨을 조절하자. 수지 (권장) 즉시 사용할 준비가 또는 냉동하지 않고, 4 ° C에 저장할 수 있습니다. </li></ol> 2. 교차 연결된 연쇄상-Tactin 폴리 메타 크릴 레이트 수지에 트윈 연쇄상 구균 - 태그 미끼 단백질 및 관련 단지의 바인딩 <ol><li> 4 ° C에서 10 분 동안 17,000 XG에서 버퍼를 결합하고 새로운 튜브에 뜨는을 전송의 미끼 단백질 시료의 원심 분리기 1 ML. </li><li> , 패 혈성-Tactin 수지에 내인성 바이오틴 단백질의 결합을 최소화 100 μg / ML의 최종 농도에 아비딘을 추가하고 4 ℃에서 15 분 동안 회전하려면 <ol><li> 단계 1.10에서 가교 수지는 4 ° C에서 장기간 보관 한 경우에, FO 400 XG에서 원심 분리에 의해 수지를 상환4 ° C에서 R 1 분, 상층 액을 버리고 TBST 1 ㎖​​로 수지를 세척한다. 원심 분리를 반복 세척 먼저 TBST로 다음 TBS와 두 번 더, 단계를 반복합니다. 4 ° C에서 1 분 400 XG에 튜브를 원심 분리기 및 초과 TBS를 제거하여 원래 볼륨을 조정합니다. </li></ol></li><li> 볼 텍싱에 의해 가교 된 패 혈성-Tactin 수지에 resuspend. 즉시 컷 피펫 팁을 사용하여 미끼 단백질 샘플이 들어있는 튜브에 수지 현탁액의 50 μl를 추가하고 4 ℃에서 또 다른 30 ​​분 동안 회전 </li><li> 기다리는 동안, 55 ° C에 thermomixer을 설정하고 단계 3.1에서 사용하기 위해 용출 버퍼 500 μl를 예열. </li><li> 4 ℃에서 1 분 400 XG에 원심 분리기 뜨는을 취소하고 미리 냉각 세척 버퍼 1 중 1 ㎖를 4 ° C에서 5 분 동안 회전에 수지를 씻어. 원심 분리를 반복하고 단계를 세 번 씻는다. 마지막 단계에서, 세척 버퍼 # 2 250 μL의 수지를 재현 탁. </li><li> TR<html> 신선한 스핀 열 수지 현탁액을 ansfer. 세척 버퍼 # 2의 또 다른 250 μL의 튜브에 남아있는 수지를 재현 탁하고 같은 C 럼에 전송할 수 있습니다. </li><li> 4 ° C에서 3 분 400 XG에 원심 분리기, 흐름을 통해 폐기하고 신선한 돌고래 코 2 ㎖ 튜브에 열을 전송합니다. 아래의 용출 단계로 바로 진행합니다. </li></ol> 특정 단백질 복합체의 3. 용출 <ol><li> 스핀 컬럼에 2.4 단계에서 예열 된 용출 버퍼 150 μl를 추가합니다. </li><li> 1,400 rpm으로 흔들어, 55 ° C에서 5 분 thermomixer에 품어. </li><li> 실온에서 1 분 1,500 XG에 원심 분리기. </li><li> 열을 버리고 ≤ -20 ℃에서 정제 된 표적 단백질을 저장 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="598"><tbody><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 인산염 완충 생리 식염수 (PBS) </td></tr>0 &quot;&gt; <td height="20" style="height:20px;width:299px;"> 나 2 HPO 4 </td><td style="width:299px;"> 10 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KH 2 PO 4 </td><td style="width:299px;"> 2mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td><td style="width:299px;"> 137 밀리 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 의 KCl </td><td style="width:299px;"> 2.7 mM의 </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> (섹션 1.4의 pH 8.0) 달리 명시되지 않는 pH는 7.4로 조정 </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 트리스 (TBS)를 완충 식염수 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH 7.4의 </td><td style="width:299px;"> 50 mM의 </td></tr><tr height="20"><td hei<html> GHT = &quot;20&quot;스타일 = &quot;높이 : 20px의 폭 : 299px;&quot;&gt; 염화나트륨 </td><td style="width:299px;"> 150 밀리미터 </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 트리스는 트윈 20 (TBST)와 완충 식염수 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH 7.4의 </td><td style="width:299px;"> 50 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td><td style="width:299px;"> 150 밀리미터 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트윈 20 </td><td style="width:299px;"> 0.1 % (V / V) </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 바인딩 버퍼 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH를 8.0 </td><td style="width:299px;"> 25 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td>X; &quot;&gt; 550 밀리미터 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 불화 나트륨 </td><td style="width:299px;"> 5 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0.5 ㎜ </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 글리세린 </td><td style="width:299px;"> 10 % (V / V) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0.1 ㎜ </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 버퍼 # 1을 씻으 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH를 8.0 </td><td style="width:299px;"> 25 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td><td style="width:299px;"> 500 밀리미터 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 불화 나트륨 </<html> TD&gt; <td style="width:299px;"> 5 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0.4 ㎜ </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 이게 팔 CA-630 </td><td style="width:299px;"> 0.2 % (V / V) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 글리세린 </td><td style="width:299px;"> 5 % (V / V) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0.1 ㎜ </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 버퍼 # 2를 세척 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH를 8.0 </td><td style="width:299px;"> 25 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td><td style="width:299px;"> 150 밀리미터 </td></tr><tr height="20">2 &quot;높이 =&quot;20 &quot;스타일 =&quot;높이 : 20px의 폭 : 597px; &quot;&gt; 용출 버퍼 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH를 8.0 </td><td style="width:299px;"> 25 mM의 </td></tr><tr height="22"><td height="22" style="height:22px;width:299px;"> SDS </td><td style="width:299px;"> 1 % (W / V) </td></tr></tbody></table> 표 1. 본 연구에 사용 된 버퍼.

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관심 없음 충돌 선언하지 않습니다.

상기 프로토콜은 비오틴 또는 강한 변성제를 포함하지 않는 임의의 적절한 버퍼에 대한 관심 및 관련 착체의 트윈-Strep의 태그 미끼 단백질을 정제하는데 사용될 수있다. 프로토콜의 현재 버전에서, 결합 및 세척이 높은 염과 비 이온 세제의 존재하에 비교적 엄격한 조건하에 수행된다. 이 적은 배경을 초래하지만, 깨지기 쉬운 단백질 복합체는 이러한 조건에서 해리 수 있습니다. 이러한 낮은 친?...

최근 몇 년 동안, 패 혈성 태그 기술은 단백질 체학 및 구조 생물학을 포함한 생물 의학 연구의 많은 분야에서 널리 사용되고있다. 짧은 Strep의 태그 펩티드로 재조합 단백질의 융합에 의존이 단백질 정제 기술은, 패 혈성-Tactin 향상된 펩티드 결합능과 스트렙 타비 딘의 유전 공학적 변형을 운반 궁합 행렬들의 출현으로 성숙. 1,2 트윈 패 혈성 태그 또는 SIII 태그, 전시 재조합 단백질을보다 효율적으로 정화를 보장, 단 하나의 패 혈성 태그를 포함하는보다 연쇄상 구균 - Tactin 행렬에 대한 높은 선호도와 지정 패 혈성 태그 II의 두 복사본을 포함하는 융합 단백질 연관된 바인딩 파트너. 그러나, 패 혈성-Tactin에 트윈 연쇄상 구균 - 태그 단백질의 높은 친화력은 또한 단점이있다. 과량의 비타민 B 복합체와 단백질의 경쟁 용출 대상 단백질 수율 감소로 이어지는, 불완전 할 수 있습니다. M광석 효율적인 대안 SDS와 용출이지만, 프로테오믹스 분석 분석 호환되지 수지에서 발표 연쇄상-Tactin와 바람직하지 않은 샘플의 오염으로 이어집니다. 이 논문은 제 화학적 가교에 의하여 패 혈성-Tactin의 수지 - 결합 사량을 안정화 한 후 얻어진 가교 수지와 트윈 연쇄상-태그 된 단백질과 관련 복합체를 용출 SDS를 사용하여이 제한을 극복하는 기술을 제시한다. 따라서, 충분한 단백질 수율함으로써 질량 분석에 의한 추가 분석을 허용 연쇄상-Tactin 샘플 오염없이 달성 될 수있다. 이 방법은 표면에 노출 된 SIII 태그 3 트윈 패 혈성 태그 (아미노산 서열 WSHPQFEK (GGGS) 각각 3 WSHPQFEK 및 SAWSHPQFEK (GGGS) 2 GGSAWSHPQFEK) 어떤 재조합 융합 단백질의 정제를 위해 적당하다. 단백질은 동물, 식물 또는 세균 기원 일 수 있으며 전체 셀 중 하나로부터 단리 될 수있다해물이나 농축 세포 기관의 분수. 예를 들어, 우리는 여기에 PVA에 감염된는 담배 benthamiana 식물의 핵 부분에서 감자 바이러스 A (PVA) 4 SIII 태그가 단백질 VPG-PRO의 정화에 대해 설명합니다. 이전에 다음과 같이 수정하여, 5 바와 같이 핵 분획을 단리 하였다 : 세포를 포름 알데히드로 처리되지 않은, 나트륨 부티레이트를 5 mM의 불소화 나트륨으로 모든 버퍼에 치환하여, 완전 프로테아제 억제제 PMSF로 치환하고, 추출 트리톤 X-100 농도 버퍼 # 2는 0.3 %로 감소 하였다 (V / V) 및 (추출 버퍼 # 3) 자당 쿠션을 통해 원심 분리하여 얻은 핵 펠릿 사전 냉장 바인딩 버퍼의 1.45 ㎖에 재현 탁하고, 4 ℃에서 1.5 시간 동안 회전 SIII-태그 미끼 단백질과 관련된 단지 (미끼 단백질 샘플)를 포함하는 결과 핵 추출물 (섹션 2 참조) 아래에 설명 된 프로토콜에 따라 처리되었습니다.

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr>
			<td>Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2mg</td>
			<td>Pierce/Thermo Scientific</td>
			<td>21585</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.</a></td>
		</tr>
		<tr>
			<td>Strep-Tactin MacroPrep resin (50% suspension)</td>
			<td>IBA</td>
			<td>2-1505</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 &#956;m, non-sterile</td>
			<td>Costar (Corning)</td>
			<td>8163</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Dolphin-nose tubes</td>
			<td>Costar (Corning)</td>
			<td>3213</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Avidin&#160;</td>
			<td>IBA</td>
			<td>2-0204</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
	</tbody>
</table>

one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)

설계 스트렙 타비 딘 (연쇄상-Tactin)를 운반하는 수지에 패 혈성 태그가 융합 단백질의 친 화성 정제는 생리 학적 조건에서 단백질 복합체의 분리를위한 널리 사용되는 방법이되고있다. 트윈 패 혈성 태그 또는 SIII 태그를 지정 패 혈성 태그 II의 두 복사본을 포함하는 융합 단백질 따라서보다 효율적인 단백질 정제를 허용, 단 하나의 패 혈성 태그를 포함하는 사람에 비해 연쇄상-Tactin에 대한 높은 친 화성의 장점이 있습니다. 그러나, 이러한 장점은 비오틴과 트윈 연쇄상 구균 - 태그 단백질의 용출이 낮은 단백질 복구에 이르는, 불완전 할 수 있다는 사실에 의해 상쇄된다. 복구 극적 도데 실 황산나트륨 (SDS)으로 변성 용출을 사용함으로써 개선 될 수 있지만 하류 단백질체 분석으로 분석 호환되지 수지로부터 방출 연쇄상-Tactin 오염을 샘플링 리드. 이 한계를 극복하기 위해, 우리는 방법 연쇄상-Tactin의 그것에 수지 결합 사량을 개발했습니다제 공유 비스 (술포 숙신 이미 딜) 수베 레이트 (BS3)와 가교하고, 생성 된 가교 된 수지를 다음 번의 배치 정제 단계에서 목적 단백질 복합체를 정제하는 데 사용함으로써 안정화된다. 패 혈성-Tactin 오염의 부재는 질량 분석에 의한 하류 단백질 분석을 허용하는 동안 SDS와 효율적인 용출, 좋은 단백질 복구를 보장합니다. 개념의 증거로서, 우리는 바이러스에 감염된 N.의 핵에서 VPG-프로 SIII 태그가 바이러스 성 단백질의 정제를 위해 여기 프로토콜을 설명 BS3으로 가교 패 혈성-Tactin의 폴리 메타 크릴 레이트 수지를 사용한 benthamiana 식물. 동일한 프로토콜은 또한 어떤 트윈 연쇄상-태그 단백질을 정제 및 생리 바인딩 파트너를 특성화하는데 사용될 수있다.

정화 절차를 개략적으로 서로 다른 기존의 정제 방법과 관련된 문제의 표현과 함께,도 1에 도시되어있다. <img alt="그림 1" fo:content-width="5.5in" src="/files/ftp_upload/51536/51536fig1highres.jpg" width="650" /> 그림 1. 정화 절차의 도식 표현. 워크 플로는 공유가 가교 수지에 트윈 연쇄상 구균 - 태그 미끼 단백질과 연관된 복합체의 결합...

Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

방법은 (연쇄상 구균 - Tactin) 수지가 공유 비스 (술포 숙신 이미 딜) 수베 (BS3)와 가교 수정 스트렙 타비 딘에 트윈 패 혈성 태그가 융합 단백질 및 특정 단지의 효율적인 정제에 설명되어 있습니다. 방법은 빠른 속도, 좋은 표적 단백질의 회수 및 고순도의 이점을 갖고, 질량 분석법에 의한 후속 분석과 호환된다.

한 단계 연쇄상-Tactin 수지 비스 (술포 숙신 이미 딜)로 가교 수베 (BS3)에 트윈 연쇄상 구균 - 태그 단백질과 그들의 단지의 정화

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

one-step-purification-twin-strep-tagged-proteins-their-complexes-on

Research

JoVE Journal

Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

18.6K 조회수.  University of Helsinki.  방법은 (연쇄상 구균 - Tactin) 수지가 공유 비스 (술포 숙신 이미 딜) 수베 (BS3)와 가교 수정 스트렙 타비 딘에 트윈 패 혈성 태그가 융합 단백질 및 특정 단지의 효율적인 정제에 설명되어 있습니다. 방법은 빠른 속도, 좋은 표적 단백질의 회수 및 고순도의 이점을 갖고, 질량 분석법에 의한 후속 분석과 호환된다. 

비디오: One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

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 ...

연구

생물학

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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 ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

인간의 DNA 수리 단백질의 유전자 연구는 모델 시스템으로 효모를 사용하여

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

한 단계 Metabolomics : 탄수화물, 단일 절차 계량 유기 및 아미노산

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

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 ...

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 ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

세포 생존에 박테리아와 그 영향의 동결 건조 공법

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

선호도 태그 재조합 단백질의 높은 처리량 정화

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

성적 증명서와 Metabolites에서의 정화<em&gt; Drosophila</em&gt; 헤드

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...