We thank Dr. Oliver Kerscher for support and advice and members of the Basrai laboratory for their support and comments on the paper. This work was supported by the National Institutes of Health Intramural Research Program.

효모 세포의 성장 (1) <ol><li> 작은 플라스크에 YPD (표 1) 30 ㎖에 효모 세포를 접종한다. 진탕 30 ℃에서 밤새 품어. </li><li> YPD 50ml에 600 나노 미터 (600 OD = 0.2)에서 0.2의 광학 밀도로 세포를 희석하고, 진탕하면서 30 ℃에서 배양한다. </li><li> 600 nm의 (OD 600 = 1.0)에서 1.0의 광학 밀도 문화를 성장. </li><li> 원심 분리기 세포 2,000 XG에 5 분 동안 상층 액을 버린다. </li><li> 세포를 세척 2000 XG에서 5 분 동안 40 ml의 멸균 원심에서 세포 펠렛을 재현 탁. 뜨는을 취소하고 -20 ℃에서 세포 펠렛을 저장합니다. </li></ol> 단백질 2. 추출 <ol><li> 또는 니켈의 superflow NTA 비드를 사용 풀다운 분석 용 빙냉 구아니딘 완충액 (표 1) 0.5 ㎖ 중의 재현 탁 세포 면역에 대한 A (표 1) 버퍼. 항상 얼음에 튜브를 유지합니다. </li><li> 2m로 이동L 스크류 캡 튜브. </li><li> 유리 구슬의 동일한 볼륨 (자료 표)를 추가합니다. </li><li> 다음 2 ~ 3 분 동안 얼음에 배치, 실온에서 2 분 동안 미니 비드 비터의 셀 (자료 표)를 구슬은-이겼다. 이 세 번 반복합니다. </li><li> 30 ~ 60 분 동안 4 ℃에서 고속에 소용돌이. 세포가 용해되어 있는지 확인하기 위해 현미경으로 시각화하여 세포를 확인합니다. 참고 : 용균 세포는 어두운 유령을 표시하고 경계 또는 정의 된 모양이 부족합니다. 최적의 셀의 적어도 80 %를 용해한다. </li><li> 15ml의 원뿔형 튜브 (스크류 캡이 느슨 함) 컬렉션에 푸시 핀과 장소를 이용하여 튜브의 바닥에 구멍을 천공. </li><li> 해물를 수집하기 위해 1 분 동안 1,000 XG에 원심 분리기. </li><li> 마이크로 원심 분리기 튜브에 해물을 전송합니다. </li><li> 4 ℃에서 30 분 동안 15,000 XG에 원심 분리기는 추출 된 단백질을 수집합니다. </li><li> ASSA 단백질을 사용하여 추출 된 단백질의 농도를 측정Y 키트 (자재 표)과 모든 추출물을 정상화는 동일한 양의 단백질을 함유한다. 적절한 버퍼 1 ml의에 총 볼륨을 가져 오십시오. 참고 : 총 단백질의 약 5 mg의 50 OD 600 세포로부터 얻어진다. </li><li> 전체 세포 추출물 (WCE)와 같은 (2.10 단계에서 추출 된 총 단백질은 5 mg의 경우 250 μg의 단백질) 50 μl를 저장합니다. 배 램 믈리 시료 완충액 (표 1) 50 μl를 첨가하고 3-5 분 동안 가열 블록에서 100 ° C에서 배양한다. 로드 웨스턴 블롯 분석 (5 절 : 웨스턴 블롯 분석)에 대한 SDS-PAGE 젤의 각 샘플의 10 μL. </li></ol> HF-Smt3 복합체 3. 정제 <ol><li> 실험 1 분간 저속 원심 분리 (800-1,500 XG)에 의해 (샘플 당 구슬 100 ㎕)에 필요한 니켈 NTA의 superflow 비즈 (재료 표)를 얻습니다. 상층 액을 제거합니다. </li><li> 워시 비즈 PBS (자료 표)의 1 ml의 5 배 : 반전 최고의 오버구슬이 재현 탁 될 때까지 바닥, 저속 원심 분리하여 구슬을 수집하고 뜨는을 제거합니다. </li><li> 구아니딘 완충액 (표 1) 1 ㎖에 비드를 일시 및 처리 대상에 대응하는 샘플 튜브의 개수로 나누어지는. 저속 원심 분리하여 구슬을 수집하고 뜨는을 제거합니다. 상단을 통해 반전 바닥에서 잘 구슬을 섞는다. 참고 : 상단 오버 바닥을 반전 잘 구슬을 섞는다. </li><li> 니켈 NTA의 superflow 구슬 100 μL에 각 샘플의 경우, (단백질의 추출 단계 2.11에서) 나머지 WCE의 950 μl를 추가합니다. </li><li> 적어도 4 시간 또는 밤새 4 ℃에서 흔들 플랫폼에 품어. </li><li> 4 ℃에서 1 분 800-1,500 XG에 원심 분리기. </li><li> 상층 액 (삭제) 50 μl를 저장합니다. 배 램 믈리 시료 완충액 50 μl를 첨가하고 3-5 분 동안 가열 블록에서 100 ° C에서 배양한다. 로드 웨스턴 블롯 분석 (제 5 SDS-PAGE 젤의 각 샘플의 10 μL : 웨스턴 블롯 항문이 Analysis). </li><li> 락을 플랫폼에 한 번 5 ~ 10 분 동안 구아니딘 버퍼 1 ml의 씻을 구슬. </li><li> 워시 비즈 락 플랫폼에서 5 ~ 10 분 동안 버퍼 (표 1)을 깨고 1 ml의 5 배. </li><li> 배 램 믈리 샘플 버퍼의 90 μL에 재현 탁 구슬. </li><li> 1 M 이미 다졸의 10 μl를 추가합니다. 참고 : 이미 다졸 인해 이미 다졸과 구슬 사이의 경쟁의 상호 작용에 니켈 NTA 비즈에서 그의 태그가 단백질을 해리하는 데 도움이됩니다. </li><li> 3-5 분간 가열 블록에서 100 ° C에서 인큐베이션. </li><li> 소용돌이, 다음 30 초 동안 13,000 XG에 원심 분리기. 새로운 튜브에 뜨는을 전송합니다. </li><li> (: 웨스턴 블롯 분석 제 5 절) 웨스턴 블롯 분석을위한 SDS-PAGE 젤에서 각 샘플의 부하 10 ~ 20 μL. 주 : WCE 5mg을 사용하는 경우는 10 ~ 20 μL, 0.5-1.0 mg의 입력에 대응한다. </li></ol> Myc와 태그가 동원체 단백질 4. 면역 침전 <ol><li> 반 C-Myc와 아가 로스 친 화성 젤을 구하는tibody (자료 표) 저속 원심 분리 (800-1,500 XG)에 의한 실험 (샘플 당 수지 25 μL)에 필요한. 상층 액을 제거합니다. </li><li> 버퍼 1 ml의 씻을 수지 5 배 : 수지가 재현 탁 될 때까지 반전 최고의 오버 바닥, 저속 원심 분리하여 수지를 수집하고, 상층 액을 제거합니다. </li><li> 완충액 1 ㎖에서 수지를 일시 및 처리 대상에 대응하는 샘플 튜브의 개수로 나누어지는. 저속 원심 분리하여 수지를 수집하고, 상층 액을 제거합니다. 참고 : 위 이상 바닥을 반전 아니라 수지를 섞는다. </li><li> 수지의 25 μL에 : (단백질의 추출 단계 2.11에서) 나머지 WCE의 950 μl를 추가합니다. </li><li> 4 ° C에서 하룻밤 흔들 플랫폼에 품어. </li><li> 4 ℃에서 1 분 800-1,500 XG에 원심 분리기. </li><li> 상층 액 (삭제) 50 μl를 저장합니다. 배 램 믈리 시료 완충액 50 μl를 첨가하고 3-5 분 동안 가열 블록에서 100 ° C에서 배양한다. 소호광고 웨스턴 블롯 분석을위한 SDS-PAGE 젤 (5 절 : 웨스턴 블롯 분석)에서 각 샘플의 10 μL. </li><li> 버퍼 1 ml의 씻을 수지 5 배 : 수지가 재현 탁 될 때까지 반전 최고의 오버 바닥, 저속 원심 분리하여 수지를 수집하고, 상층 액을 제거합니다. </li><li> SUMEB 샘플 완충액 (표 1) 100 ㎕에 재현 탁 수지. 참고 : SUMEB 샘플 버퍼가 8 M 우레아 1 % SDS (강한 변성 조건)가 포함되어 있습니다. </li><li> 3-5 분간 가열 블록에서 100 ° C에서 인큐베이션. </li><li> 소용돌이, 다음 30 초 동안 13,000 XG에 원심 분리기. 새로운 튜브에 뜨는을 전송합니다. </li><li> (: 웨스턴 블롯 분석 제 5 절) 웨스턴 블롯 분석을위한 SDS-PAGE 젤에서 각 샘플의 부하 10 ~ 20 μL. 주 : WCE 5mg을 사용하는 경우는 10 ~ 20 μL, 0.5-1.0 mg의 입력에 대응한다. </li></ol> 5. 웨스턴 블롯 분석 <ol><li> 4~12% 비스 - 트리스 젤 (자료 표)와 반환 한의 단백질 추출물을로드ORM 전기는 90 분 동안 120 V에서 (버퍼를 실행 : 자료 표). </li><li> 90 분 (: 자재 표 전송 버퍼)에 대해 30 V로 전송 장치를 이용하여 니트로 셀룰로오스 막 (자재 표)로 겔로부터 단백질을 옮긴다. </li><li> 실온에서 1 시간 동안 5 % 우유 / 1X TBST (표 1)에서 막을 차단. </li><li> 5 % 우유 / 1X TBST 밤새 4 ° C가 차 항체 (자료 표)와 멤브레인을 품어. 1,000 (안티 - 플래그 및 안티 UB 항체) 또는 1 : 5,000 (안티 - Myc와 항체) 일차 항체를 들어, 1의 희석을 사용합니다. </li><li> 1X TBST 10 분마다 막 배를 씻으십시오. </li><li> 실온에서 1 시간 동안 5 % 우유 / 1X TBST에서 이차 항체 (자재 도표)와 함께 막을 인큐베이션. 이차 항체의 5,000 희석 : 1을 사용합니다. </li><li> 1X TBST 10 분마다 막 배를 씻으십시오. </li><li> ECL 가공용으로 멤브레인을 품어NG 용액 (자료 표) 5 분. </li><li> 블루 민감한 X 선 필름 막 (자료 표)를 노출 및 자동 개발자 (자료 표)를 사용하여 개발할 수 있습니다. </li></ol>

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The authors have nothing to disclose.

이러한 HA, Myc와, 플래그 및 GST와 같은 에피토프 태그 널리 단백질의 생화학 적 분석을 위해 사용된다. HF-Smt3 및 Ndc10 Ndc80과 같은 나 Myc 태그가 동원체 단백질과 균주의 구성은 이러한 sumoylation 유비퀴틴과 같은 PTMS의 검출을 용이하게한다. HF-Smt3 분석를 풀다운는 sumoylated 동원체 단백질, Ndc10 및 Ndc80 (그림 1)의 검출을 할 수 있습니다. 안티 - 플래그 항체를 이용하여 친 화성 정화 프로토?...

각각 표적 단백질에 (S. cerevisiae의 1 Smt3 SUMO) 유비퀴틴과 sumoylation는 유비퀴틴과 작은 유비퀴틴 같은 수정의 활용을 할 수 있습니다. 동원체 단백질 PTMS 충실한 염색체 분리를 보장하기 위해 상이한 세포주기 단계에서 자신의 세포 수준 및 단백질 - 단백질 상호 작용에 영향을 미친다. 예를 들어, Cse4 / CENP-A 및 외부 동원체 단백질 Dsn1의 세포 수준 게놈 안정성 2-5을 보장하기위한 유비퀴틴 매개 단백질 분해에 의해 조절된다. 잘못된 동원체 - 미세 소관 첨부 불안정 직접 미세 소관 6-8과 상호 작용 Dam1과 Ndc80 단지를 인산화 Ipl1 / 오로라 B 키나제가 필요합니다. 일흔을 통해 동원체 단백질의 확인에도 불구하고, PTMS, 예를 들어, 유비퀴틴과 SUMO 이러한 단백질의 변형을 조사 거의 연구가있다. 주요한 제한은 PTM을 보존하는 능력이다정화 및 sumoylation, 인산화, 메틸화, 등과 같은 PTMS의 검출을위한 사용자 정의 항체의 소수 중의. sumoylated 동원체 단백질의 특성은 Ndc10, Cep3는 Bir1 및 Ndc80 사용자 정의 항체 (9)를 이용했다. 또한 Ndc10 유비퀴틴은 10 기재로서 연루되어왔다. 인간 Hec1 (S. cerevisiae의에서 Ndc80)도 APC / C-hCdh1 E3 리가 아제 (11)에 의해 규제, 유비퀴틴 화를 위해 기판이다. 따라서, Ndc10 및 Ndc80는 S.에서 sumoylation 및 유비퀴틴을 모두 감지 할 수있는 프로토콜의 최적화를위한 좋은 후보입니다 cerevisiae의. sumoylation의 식별을 용이하게하기 위해, 우리는 HF-Smt3 및 Myc와 태그가 Ndc10 또는 Ndc80을 표현 균주를 건설했다. 에피토프 태그 (HF :을 His6-FLAG)의 사용으로 인해 자주 후보 단백질에 대해 발생하는 폴리 클로 날 혈청에서 관찰되고 교차 반응의 배경을 최소화한다. 우리는 선호도와 프로토콜을 고안HF-Smt3 접합체를 정제 한 다음 정제 Smt3 제제 sumolyated Ndc10 및 Ndc80의 존재를 검출하기 위해 상업적 방지 플래그 및 안티 나 Myc 항체를 사용 하였다. 유비퀴틴을 위해, 우리는 Myc와 태그가 동원체 단백질의 유비퀴틴 화를 보존하는 변형 된 면역 프로토콜을 고안 Ndc10과 Ndc80의 유비퀴틴 화를 감지하는 상업 항 UB 항체로 웨스턴 블롯 분석을 수행 하였다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass beads</td>
			<td>BioSpec Products</td>
			<td align="right">11079105</td>
			<td>0.5 mm dia.</td>
		</tr>
		<tr>
			<td>Mini beadbeater</td>
			<td>BioSpec Products</td>
			<td>#693</td>
			<td>8 cell disrupter</td>
		</tr>
		<tr>
			<td>Ni-NTA superflow</td>
			<td>Qiagen</td>
			<td align="right">30430</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8215</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Anti-c-Myc agarose affinity gel antbody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>A7470</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Monoclonal anti-Flag M2 antibody produced in mouse</td>
			<td>Sigma-Aldrich</td>
			<td>F1804</td>
			<td>Primary antibody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>c-Myc antibody (A-14)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-789</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Purified mouse antibody monoclonal 9E10</td>
			<td>Covance</td>
			<td>MMS-150P</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Ubiquitin (P4G7) monoclonal antibody</td>
			<td>Covance</td>
			<td>MMS-258R</td>
			<td>Primary antibody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>ECL Rabbit IgG, HRP-linked whole Ab</td>
			<td>GE Healthcare Life Sciences</td>
			<td>NA934V</td>
			<td>Secondary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>ECL Mouse IgG, HRP-Linked Whole Ab</td>
			<td>GE Healthcare Life Sciences</td>
			<td>NA931V</td>
			<td>Secondary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>DC protein assay</td>
			<td>Bio-Rad</td>
			<td>500-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Novex</td>
			<td>LC2001</td>
			<td>0.45 mm pore size</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>Novex</td>
			<td>NP0321BOX</td>
			<td>1.0 mm, 10 well</td>
		</tr>
		<tr>
			<td>NuPAGE MES SDS Running Buffer</td>
			<td>Novex</td>
			<td>NP0002</td>
			<td>20x</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Novex</td>
			<td>NP0006-1</td>
			<td>20x</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td align="right">34078</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS pH7.4</td>
			<td>GIBCO</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue sensitive X-Ray film</td>
			<td>Dbio</td>
			<td>DBOF30003</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic developer</td>
			<td>Kodak</td>
			<td>M35AX-OMAT</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>MG-132</td>
			<td>Selleck Chemicals</td>
			<td>S2619</td>
			<td>25 mg</td>
		</tr>
	</tbody>
</table>

protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80

이러한 인산화, 메틸화, 아세틸 화, 유비퀴틴 및 sumoylation 같은 번역 후 변형 (PTMS)는, 많은 단백질의 세포 기능을 조절한다. 센트로 DNA와 연관 동원체 단백질의 PTMS는 유전체의 안정성을 유지하기 위해 충실한 염색체 분리를 중재. 같은 질량 분석 및 웨스턴 블롯 분석과 같은 생화학 적 접근 방법이 가장 일반적으로 PTMS의 식별을 위해 사용된다. 여기서, 단백질의 정제 방법은, 사카로 마이 세스 세레 비지에 동원체 단백질 Ndc10 및 Ndc80의 sumoylation 및 유비퀴틴 양쪽의 검출을 허용하는 기술된다. Ndc10 Myc와 - 태그 또는 Ndc80가 건설하고 우리의 연구에 사용 된 표현 폴리 히스티딘 플래그 태그가 Smt3 (HF-Smt3) 및 변형. sumoylation의 검출을 위해, 우리는 니켈 비드를 사용하여 자신 태깅 sumoylated 단백질을 정제 친화력 프로토콜을 고안 sumoylated Ndc10 A를 검출하기 위해 항 - 나 Myc 항체로 웨스턴 블롯 분석을 이용ND Ndc80. 유비퀴틴의 검출을 위해, 우리는 Myc와 태그가 단백질의 면역을위한 프로토콜을 고안 Ndc10과 Ndc80이가 도처 것을 보여 방지 UB 항체와 웨스턴 블롯 분석을 사용했다. 우리의 결과는 그의 플래그에 대한 관심의 에피토프 태그 단백질이 Smt3 변형 여러 PTMS의 검출을 용이하게 태그를 보여줍니다. 앞으로의 연구는이 기술의 개발은 특정 PTM에 의존 단백질 상호 작용을 식별하고 특성화 할 수 있도록해야한다.

이전에 설명 된대로 9,12- 동원체 단백질과 Ndc80 Ndc10의 sumoylation을 검출하도록, HF-Myc와 Smt3 및 태깅 동원체 단백질 (또는 Ndc80 Ndc10)와 균주 (표 2)를 구성 하였다. HF-Smt3 접합체 친화력의 Ni-NTA 비드를 사용하여 정제 하였다. HF-Smt3 (그림 1A와 1B, 왼쪽 패널)없이 제어 변형에 결석했다 스모 기판의 sumoylated 형태의 안티 - 플래그 항체 허용 감지와 정제 HF-Smt3의 웨?...

Watch this Scientific Journal Video about Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 at JoVE.com

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

이 원고는 신진 효모 사카로 마이 세스 세레 비지에, sumoylation 및 동원체 단백질, Ndc10 및 Ndc80의 유비퀴틴의 검출에 대해 설명합니다.

Sumoylation과 신진 효모 동원체 단백질 Ndc10과 Ndc80의 유비퀴틴 화를 감지 할 수 있습니다 단백질 정제 기술

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

protein-purification-technique-that-allows-detection-sumoylation

Research

JoVE Journal

Biology

12.0K 조회수.  National Cancer Institute, National Institute of Health.  이 원고는 신진 효모 사카로 마이 세스 세레 비지에, sumoylation 및 동원체 단백질, Ndc10 및 Ndc80의 유비퀴틴의 검출에 대해 설명합니다. 

비디오: Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

버딩 효모에 Replicative 수명 측정

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

연구

생물학

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

단백질 Ubiquitination의 감지

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

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 ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

효모 세포 Mitochondria의 정화

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

버딩 효모에서 단백질 농도의 함수로 형태학의 표현형의 발전 분석

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

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.  ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

신진 효모의 형광 시간 경과 영화를 획득 및 이식을 사용하여 단일 세포 역학 분석

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

함수의 연구, 상호 작용, 그리고 번역 후 수정을위한 신진 효모 단백질 추출 및 정제

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

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 ...