Work in the authors&#39; laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

1 세대와 전체 길이 또는 FLAG의 돌연변이 버전 표현 HEK293 안정 세포주의 문화 에피토프를 - 태그 Ino80 또는 기타 INO80 복잡한 서브 유닛 <ol><li> 전체 길이 또는 돌연변이 인간 Ino80 ATPase의 또는의 프레임, N 단자 FLAG 에피토프 태그 포유 동물 발현 벡터 pcDNA5 / FRT로 다른 INO80 서브 유닛을 코딩하는 cDNA 클론. </li><li> 진행하기 전에 DNA 시퀀싱에 의해 삽입 된 cDNA의 서열을 확인한다. </li><li> 형질 감염을 수행하려면, DMEM (둘 베코 변형 이글 중간), 5 %의 글루타민을 함유하는 배지에서 10cm 조직 배양 접시에 HEK293 세포에서 FLP을 성장에, 10 % FBS (소 태아 혈청). </li><li> 세포 ~ 70 % 컨 플루 도달하면, 전체 부피에서, 각각의 조직 배양 접시에 FuGENE6 형질 감염 시약 40 μL, 적절한 pcDNA5 / FRT 발현 플라스미드 0.5 μg 및 FLP 재조합 효소를 암호화 pOG44, 9.5 μg의 혼합물을 추가 800 μL의. </li><li> 48 시간 후,를 Trypsinize와의PLIT 1시 반 (10)에 접시 형상의 비율로 세포를, 5 % 글루타민, 10 % FBS DMEM으로 그들을 성장하고, 3, 100 μg / ㎖ 하이 그로 마이신 B - 4 주. (- 5 일 일반적으로 매 3) 노란색으로 시작 때마다 배지를 변경합니다. </li><li> FLAG-태그 된 단백질의 최고 레벨을 표현하는 포지티브 클론을 확인하기 위해, 개별적인 하이 그로 마이신 B 내성 콜로니를 선택하고, 24 웰 플레이트의 한 웰에 전송할. <ol><li> 세포를 5 분 동안 1,000 XG에서 원심 ~ 1 ml의 PBS 및 펠릿의 각 웰에서 80 % 컨 플루 수확 셀에 도달하면. </li><li> 상등액을 제거한 후, SDS-PAGE 샘플 완충액 60 μL에서 세포 펠렛을 재현 탁. </li><li> SDS 페이지와 FLAG의 발현을 모니터링하는 서양 모래 바닥에 재현 탁 세포 펠렛의 주제 절반 미끼 단백질을 태그; 향후 분석을 위해 나머지 절반을 저장합니다. </li></ol></li><li> 더 FLAG-태그 된 인간 Ino80 단백질은 세포 용 해물에서 검출되지 않으면, 초기 클론 세포를 확장 백성들이 란15cm 조직 배양 접시에 24 웰 플레이트의 한 우물에서 세포를 도금 더 만큼은. <ol><li> 15cm 요리, 50 ML 원뿔 튜브에 얼음 차가운 PBS에 합류 세포 근처에 resuspend 및 전송에서 세포를 수확합니다. </li><li> 추가 PBS를 첨가하여 50 ㎖의 최종 부피로 준비한다. </li><li> 펠릿을 5 분 동안 1,000 XG에 세포 및 상등액을 최대한 제거한다. </li><li> Lys450 완충액 1 ㎖에서 세포 펠릿 (20 mM의 HEPES-NaOH를 pH를 7.9, 450 mM의 염화나트륨을 재현 탁, 0.5 % 트리톤-100, 10 mM의 KCl을 4 mM의 MgCl2를, 0.2 mM의 EDTA, 10 % 글리세롤, 1 mM의 DTT, 200 μM의 PMSF, 1 : 1000 프로테아제 억제제 칵테일). 참고 : 여기와 다른 곳에서 항상 즉시 실험을 시작하기 전에 버퍼에 DTT, PMSF 및 프로테아제 억제제 칵테일을 추가합니다. </li><li> 면역 침전 방지 FLAG 아가로 오스 겔의 20 μl를 사용하여 결과 전체 세포 용 해물에서 단백질을 FLAG는 - 태그 및 서부 블로 팅에 의해 FLAG 용출액을 분석 할 수 있습니다. [immun의 자세한 내용은oprecipitation 절차, 단계 5 참조] </li></ol></li><li> 원하는 클론 세포 라인 (12)의 냉동 주식을 준비하고 사용할 때까지 액체 질소에 보관합니다. </li></ol> 롤러 병에 2 성장 HEK293 세포주 INO80 단지의 대규모 준비를 위해, 배양 세포를 10 - 20 롤러 병; 각 롤러 병에서 전형적인 수율은 포장 세포의 ~ 1 ML입니다. <ol><li> 각 롤러 병으로, 하이 그로 마이신 B를하지 않고 200 ㎖의 DMEM 5 % 글루타민, 10 %의 혈청을 추가 </li><li> 각 롤러 병에 단일 근 합류 15cm 접시에서 세포를 모두 전송 </li><li> 장소 롤러 37 ° C 롤러 병 인큐베이터에 병은 0.2 rpm으로 회전합니다. </li><li> 세포 ~ 70 %의 포화 상태에 도달하면, 부어 및 매체를 폐기합니다. </li><li> 각 병에 ~ 얼음 50 ㎖의 차가운 PBS를 추가합니다. 병을 잡고 수평으로 부드럽게 세포 단일 층을 풀어 소용돌이 친다. </li><li> 250 ml의 플라 스트에 재현 탁 세포로 이동IC 원뿔 병 및 얼음에 장소. </li><li> 추가 PBS와 롤러 병을 씻어 병하는 병에서 순차적으로 전환하기. 세정액이 더이상 분명 (전형적으로는 약 5 병을 린스하기 위해 사용 된 후) 없을 때, 세포 현탁액에 추가. </li><li> 를 10 분 동안 400 XG에 세포를 원심 분리에 의해 펠렛. </li><li> 부드럽게 PBS로 세포를 재현 탁 한 250ml의 원추형 병로 결합하고, 추가 처리 될 때까지 얼음 상에 유지한다. </li></ol> 또 다른 FLAG-태그 INO80 서브 유닛을 발현하는 세포에서 INO80 서브 유닛의 3은 siRNA를 매개 넉다운 하나의 서브 유닛이 부족 INO80 서브 콤플렉스를 얻으려면 사용 FLAG-immunopurification 안정적으로 shRNA를 발현 siRNA를 처리 한 세포 또는 세포로부터 INO80 단지를 정화합니다. (작은 간섭 RNA) 형질 전환 프로토콜이 여기에 설명 된 &quot;역&quot;의 siRNA는 15cm 접시에서 성장 HEK293 세포에 최적화되어 있습니다. 프로토콜은 세포와의 소 단일 15cm 접시입니다ULD 필요한 세포의 수에 따라 적절하게 확장 할. siRNA를 처리 한 세포에서 INO80 단지의 생화학 적으로 유용한 금액을 준비하기 위해, 하나는 40 15cm 접시에서 자란 문화까지 확장한다 패킹 된 세포 펠렛을 4 ml의 -이 약 2를 수득한다. <ol><li> 안정적으로 15cm 요리에 가까운 포화 상태에 INO80 단지의 원하는 서브 유닛을 표현하는 HEK293 세포를 성장. </li><li> 의 siRNA 재현 탁 완충액의 부피 추가 (20 mM의 KCl을, 6 mM의 HEPES pH가 7.5, 0.2 mM의 MgCl2를) 충분한 동결 건조 된 siRNA를 포함하는 siRNA를 튜브의 50 μM 원액을 제조 하였다. 솔루션을 위로 피펫 몇 번 아래로 siRNA를 완전히 용해하기 위해 실온에서 30 분 동안 nutator에 품어. </li><li> siRNA를 형질 전환 및 시약을 포함하는 형질 전환 칵테일을 준비합니다. 매우 부드러운 혼합과 혈청 중 감소 32 ㎕의 리포 펙 타민 RNAiMAX로 50 μM의 siRNA 원액의 10 μl를 혼합하고 옵티-MEM의 4 ML에 추가; 알낮은 모든 시약은 사용 전에 실온으로 평형을 유지한다. </li><li> 30 분 동안 RT에서 혼합물을 배양한다. </li><li> 안정적으로 형질에 대해 원하는 INO80 서브 유닛을 표현 FLP-에서 HEK293 세포를 준비합니다. <ol><li> 단계 3.4의 배양 동안 RT PBS로 한번 15cm 접시에 세포를 씻으십시오. </li><li> 그들은 판을 들어 올릴 시작 때까지만 PBS를 제거한 후, 1 ml의 트립신으로 세포를 취급합니다. </li><li> 즉시 세포를 트립신 부드럽게 섞어 완전 배지 (DMEM + 5 % 글루타민 + 10 % FBS) 10 ㎖를 추가합니다. 실온에서 5 분 동안 1,000 XG에서 원심 분리하여 세포를 수집합니다. </li><li> ~ 4 ml의 완전 배지에서 세포 펠렛을 재현 탁하고, 혈구 계를 사용하여 셀 재 부유 카운트. </li><li> ~ 5.4 × 106 / ㎖의 농도로 완전 배지와 세포를 희석. </li></ol></li><li> 각 15cm 접시에, 4 ml의 형질 칵테일 다음 15 ㎖의 전체 문화 매체를 추가합니다. thoro있는 중간 형질 칵테일을 보장하기 위해 부드럽게 소용돌이ughly 혼합. 마지막으로, 세포를 균일하게 분산시키는 것이 부드럽게 소용돌이 다시 세포 현탁액 중 하나 mL를 넣고. </li><li> 37 ° C, 5 % CO 2 배양기에서 배양 60 시간 후, 부드럽게 매체, 얼음 차가운 PBS에 재현 탁 세포를 제거하고 핵 추출물을 준비하는 즉시 진행합니다. </li></ol> 핵 추출물 (4) 준비 이 절차 (13)의 Dignam 프로토콜에서 수정 된 세포 펠렛을 시작 크기에 따라 상하 또는 축소 될 수있다. 일반적으로, 압축 된 세포 펠렛 수율 최종 핵 추출물 1 ㎖를 1 ㎖. 모든 버퍼는 차가운 얼음해야하고, 적당한 차가운 방을 사용할 수없는 경우 모든 단계는 추운 방에서 또는 얼음에서 수행해야합니다. <ol><li> 핵의 분리 : <ol><li> 조심스럽게 4 ° C에서 10 분 동안 1,000 XG에 (15 또는 50 ml)을 적절하게 크기에 세포를 원뿔 튜브와 스핀 졸업 전송할 수 있습니다. </li><li> 상층 액을 제거하고 포장 세포 화소의 양을 측정할 수 있습니다. 포장 된 세포의 1 ㎖를 2 ~ 3 × 10 8 HEK293 세포에 해당합니다. </li><li> 5 포장 셀 버퍼의 볼륨 (1,000 프로테아제 억제제 칵테일 10 mM의 HEPES, pH가 7.9, 1.5 mM의 MgCl2를, 10 mM의 KCl을 갓 1 mM의 DTT, 200 μM의 PMSF를 첨가하고, 1)를 추가합니다. 부드럽게 피펫 팅하여 세포 펠렛을 재현 탁. </li><li> 정확히 10 분 동안 얼음에 품어. </li><li> 4 ° C에서 10 분 동안 1,000 XG에 펠렛은 세포 상층 액을 제거합니다. </li><li> 세포 펠렛의 크기는 버퍼 A.에서 배양시 2 배까지 증가해야 </li><li> 버퍼의 충전이 셀 볼륨에서 세포를 재현 탁하고, 적절한 크기의 다운스 균질 조직에 세포 현탁액을 전송. 패킹 세포 미만 2ml를 시작하면, 7 ㎖의 균질화를 사용; 2 - 4 ml의 포장 세포, 15 ㎖의 균질화를 사용; 및 포장 세포의 10 이상 ML를 들어, 40 ㎖의 균질화를 사용합니다. </li><li> 다운스 균질 유엔의 LOOSE 유리와 유 봉, 세포 현탁액을 균질화세포의 90 % 참깨 1 % 트리 판 블루로 염색 긍정적. </li><li> JA-17 또는 이와 유사한 회에서 4 ° C에서 20 분 동안 25,000 XG에 45 ㎖의 고속 원심 분리기 튜브와 스핀에 현탁액을 전송 (자재 / 장비의 표 참조). </li><li> 핵 펠렛에서 세포질 단백질이나 분별 동안 핵 밖으로 누출 단백질을 포함하는 상층 액을 제거합니다. </li></ol></li><li> 소금 핵을 추출 : <ol><li> 추가 버퍼 C (를 20mM HEPES, 산도 7.9, 25 % 글리세롤, 1.5 mM의 MgCl2를, 0.2 mM의 EDTA, 갓 추가 한 mM의 DTT, 200 μM의 PMSF, 1 : 1000 프로테아제 억제제 칵테일) 핵 펠렛에; 포장 세포 부피 (~ 1 × 109 세포를) 시작하는 매 3 ㎖의 2.5 ml의 버퍼 C를 사용합니다. </li><li> 유리로드 또는 피펫을 사용하여, 관의 벽에서 핵을 제거하고 펠릿을 적절한 크기의 다운스 균질 혼합물 전체에 옮긴다. </li><li> 번째 균질화하여 핵을 재현 탁LOOSE 유 두 스트로크 전자 혼합물. </li><li> 냉장 비커에 재현 탁 핵 부분을 전송합니다. 서스펜션 깊은 적어도 0.5 cm로 비커를 채울 것 같은 것을 비커를 선택합니다. </li><li> 부드럽게 얼음에 피펫 또는 유리 막대로 현탁액을 교반하면서 염색질 또는 다른 불용성 구조에서 핵 단백질을 추출하려면, 서서히 5 M NaCl을 적가하여 0.42 M 염화나트륨으로 서스펜션의 소금 농도를 증가; 5 M NaCl을 모두 첨가 한 후, 용액은 매우 점성 또는 겔형이 될 것이다. M의 NaCl은 다음 식에 따라 0.42 M 염화나트륨의 최종 농도로 용액을 가지고 필요한 5의 체적을 계산한다 : <img alt="식 (1)" fo:content-width="4.5in" src="/files/ftp_upload/51720/51720equation1.jpg" width="500" /></li><li> 조심스럽게 유형 45 티 O를위한 유형 70.1 티 로터에 10 ㎖의 폴리 카보네이트 튜브에 점성 현탁액을 전송 (또는 동급) 또는 70 ㎖의 폴리 카보네이트 병비슷한 로터 (자재 / 장비의 표 참조) r에. 70 ㎖의 병을 사용하는 경우 10 ㎖의 튜브를 사용하거나 캡 어셈블리 경우 파라 필름으로 단단히 밀봉합니다. </li><li> 천천히 nutator를 사용하여 30 분 동안 4 ° C에서 밀봉 된 튜브 바위. </li><li> 4 ° C에서 120,000 XG에 45 형 티 또는 30 분 동안 70.1 티 로터에 샘플을 스핀. </li><li> 하나의 플라스틱 튜브 나 병에 뜨는을 전송합니다. 이 상층 액은 핵 추출물, 그리고 펠렛 염색질 및 기타 핵 파편이 포함되어 있습니다. </li><li> 편리 크기 분취에 핵 추출물을 나누 액체 질소에 냉동 및 -80 ° C에서 보관하십시오. </li></ol></li></ol> 인간 INO80 또는 INO80 서브 콤플렉스의 5 면역 정화 <ol><li> 냉동 핵 추출물을 해동, 동결 물까지 손 사이의 벤치 탑 또는 롤 튜브 추출물을 포함하는 장소 튜브 슬러리된다. 추출물 완전히 t 될 때까지 얼음 또는 차가운 방에 튜브를 배치hawed. </li><li> 동결 - 해동 사이클 동안 형성 할 수있는 어떤 침전물을 제거하는 유형 70.1 티 로터 또는 동급에서 4 ° C에서 20 분 동안 100,000 XG에서 해동 핵 10 ㎖의 폴리 카보네이트 초 원심 분리기 튜브에 압축 스핀을 전송합니다. </li><li> 15 ML 원뿔 관에 뜨는을 전송합니다. 1000 프로테아제 억제제 칵테일 : 1 mM의 DTT, 200 μM의 PMSF, 그리고 하나의 최종 농도에 신선한 DTT, PMSF 및 프로테아제 억제제 칵테일을 추가합니다. </li><li> , immunopurification에 대한 항-FLAG 아가로 오스를 준비 끝이 깨끗한 메스로 절단되어있는 팁 P200 또는 유사한 피펫을 사용하여 1.5 ml의 microcentrifuge 관에 안티-FLAG 아가로 오스 비즈의 50 % 슬러리 200 μl를 전송하려면 또는 면도날. </li><li> 30 초 동안 8,000 XG에서 벤치 탑의 microcentrifuge에서 원심 분리하여 구슬을 펠렛. 상층 액을 제거하고, Lys450 버퍼의 1 ㎖에 구슬을 재현 탁하여 구슬을 세척하고, 30 초 동안 8,000 XG에 구슬을 펠렛. B를 세척두 번 더 EADS. </li><li> 끝이 깨끗한 메스 또는 면도날과 같은 팁을 사용, 양도와 차단되어있는 팁 P200 또는 유사한 볼륨 조절 피펫을 사용하여 핵 추출물의 약 100 μL의 세척 방지 FLAG 아가로 오스 비즈를 재현 탁 추출물을 함유하는 15 ml의 원추형 튜브에 재현 탁 구슬. 구슬의 모두 15 ML 튜브로 전송 될 때까지 몇 번을 반복합니다. </li><li> 실험실 회전에 느린 회전 4 ° C에서 4 시간 동안 추출 / 비드 혼합물을 품어. OPTIONAL : DNA 오염을 제거하기 위해 25 단위 / ml의 농도로 benzonase 포함한다. </li><li> 4 ° C에서 5 분 동안 1,000 XG에서 원심 분리 FLAG 아가로 오스를 수집합니다. </li><li> 10 ml의 Lys450에서 재현 탁은 씻어 부드러운가 nutator에 락과 4 ° C에서 5 분간 배양한다. 4 ° C에서 5 분 동안 1,000 XG에 구슬을 펠렛. </li><li> 1.5 ml의 microcentrifuge 관에 Lys450 및 전송 비즈 150 μL - (100)에서 재현 탁. 공동모든 구슬이 microcentrifuge 관에 전송 될 때까지 Lys450 150 μL - ntinue 100과 15 ML 원뿔 튜브를 세척합니다. </li><li> 마이크로 원심에서 4 ° C에서 30 초 동안 8,000 XG에 구슬을 스핀 다운. 1 ㎖의 EB100 버퍼 (10 mM의 HEPES pH가 7.9, 10 % 글리세롤, 100 mM의 염화나트륨, 1.5 mM의 MgCl2를, 0.05 %는 트리톤-100, 갓 추가 한 mM의 DTT, 200 μM의 PMSF 회 1 ml의 Lys450로 3 회 이상 세척하고 및 1 : 1000 프로테아제 억제제 칵테일). </li><li> , 결합 된 단백질을 용출 0.25 ㎎ / ㎖ 1 배 FLAG 펩타이드를 포함하는 200 μL의 EB100 버퍼를 추가합니다. nutator에 4 ° C 각각에서 30 분을 품어. </li><li> 마이크로 원심에서 4 ° C에서 30 초 동안 8,000 XG에 구슬을 펠렛. 새로운 microcentrifuge 관에 용출 INO80 복합체를 포함하는 상층 액을 전송합니다. </li><li> 용출 네 번 이상 반복 한 하나의 튜브로 모든 상층 액을 풀. </li><li> 용출 된 단백질 분획으로부터 잔여 FLAG-아가 로스 비드를 제거하려면,빈 스핀 칼럼을 통해 용출액을 전달합니다. </li><li> 용출 된 단백질 분획을 농축 ~ 10 배의 초 원심 분리 필터 장치 (50,000 분자량 컷오프)을 사용. </li><li> FLAG 펩티드를 제거하려면이 탈염 컬럼을 통해 농축 된 단백질 분획을 순차적으로 전달합니다. </li><li> 액체 질소에 냉동 및 -80 ° C에서 보관, 20 ㎕의 분취 액으로 정제, 탈염 단백질 분획을 나눈다. </li><li> 실버 스테인드 젤 또는 웨스턴 블롯에 의해 INO80 또는 INO80의 서브 콤플렉스의 서브 유닛의 구성을 분석하고 표준으로 알려진 농도의 재조합 INO80 서브 유닛의 준비를하여 반 정량적 웨스턴 블로 팅하여 농도를 추정한다. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Structural and functional studies of multi-subunit mammalian chromatin remodeling complexes from higher eukaryotes have been hampered by the difficulty of preparing biochemically useful amounts of such complexes containing mutant subunits or lacking certain subunits altogether. There are a number of technical hurdles: First, genetic manipulation in mammalian cells has been technically challenging and time-consuming. Unlike yeast cells, whose genome can be readily edited and targeted using recombineering techniques, the mammalian genome is more structurally complex and less susceptible to recombineering interventions. Thus, deletion or modification of mammalian genes in cultured cells or animals is more time consuming and requires more specialized expertise. As a consequence, the generation of mutant mammalian complexes carrying structural mutations or lacking subsets of subunit(s) has been rate-limiting. Second, biochemical reconstitution approaches using heterologous protein expression systems are often used to generate defined multi-protein assemblies. However, such approaches become technically challenging for very large complexes, whose subunits may need to be simultaneously expressed in proper stoichiometry and/or to be assembled in a particular order, with help from specific chaperones or cofactors. These problems are especially severe in the case of the INO80 complex, because its Ino80 ATPase subunit is particularly difficult to express in insect or E. coli cells in biochemically useful amounts. In studies of the INO80 chromatin remodeling complex, it has been possible to circumvent some of these challenges using the strategies described in this protocol.
Generating human cell lines stably expressing epitope tagged subunits of the INO80 complex is a key step in this procedure, as it enables the purification of well-defined chromatin remodeling complexes that can be tested using various biochemical assays. Although it is in principle more time consuming to generate stable cell lines than to use transient transfection to deliver engineered cDNAs into mammalian cell lines for the production of recombinant protein, transient transfection suffers from several drawbacks. First, DNA&#8213;in the form of a transiently transfected extra-chromosomal vector&#8213;is short-lived and usually cannot self-propagate during the cell cycle. Second, transfection efficiency varies greatly depending on cell type and growth conditions. Heterogeneous transfection can cause a mosaic expression pattern that confers a selective growth advantage or disadvantage within the cell population. Thus, transiently introduced transgenes tend to get lost during the many cell passages required to generate the large amount of cells needed for purification of biochemically useful amounts of protein. Stable cell lines are most commonly generated using methods that result in random integration of cDNAs encoding a protein of interest. However, randomly integrated cDNAs can disrupt expression of endogenous genes and are subject to gene silencing with multiple cell passages. For these reasons, we typically introduce Ino80 cDNAs into cells using Flp-mediated recombination technology, in which the cDNA is stably incorporated into a specific chromosomal location via Flp recombinase-mediated insertion into a single FRT site stably integrated into the genome.
During selection of stable cell lines, it is essential to be able to detect exogenously expressed, FLAG-tagged, wild type or mutant Ino80 proteins. Because FLAG-tagged Ino80 protein is often expressed at very low levels and is therefore difficult or impossible to detect by western blotting in crude lysates of cells grown in a single well of a 24-well plate, it is often necessary to expand the initial clonal cell population prior to screening for expression of Ino80 protein. FLAG-tagged Ino80 protein can then be purified and concentrated by FLAG immunopurification prior to analysis by Western blotting.
The efficiency of siRNA-mediated knockdown is quite sensitive to cell density at the time of transfection; we found that knockdown efficiency decreased when transfections were performed with cells at densities other than that recommended in the protocol. The optimal length of time for siRNA transfection is variable and needs to be determined empirically for each target protein, as it depends on the stability of the target protein, the turnover rate of the targeted protein in protein complexes, and the degree to which the protein is essential for cell viability. As an alternative to the use of transient transfection of siRNAs to deplete individual subunits, one may wish to consider generating cell lines stably expressing shRNAs under selective pressure as it may be easier and more cost-effective to grow biochemically useful amounts of such cell lines. However, it can prove difficult to obtain sufficient knockdown using shRNA-mediated knockdown in stable cell lines.
Also key to the success of our procedure is the use of nuclear extracts as the starting material for purification of INO80 complexes. We have found that immunopurified INO80 complexes from whole cell extracts are often contaminated with ATPase and/or nucleosome remodeling activities that are independent of the Ino80 ATPase; such contamination is largely avoided when complexes are purified from nuclear extracts.
Preparation of nuclear extracts and isolation of intact INO80 complexes depends on a number of critical factors. First, the cells used to prepare nuclear extracts should be intact and healthy. The washed cell pellet is expected to swell up to two-fold following incubation in the hypotonic Buffer A. If cells do not swell and/or the supernatant becomes turbid at this step, the starting population of cells may have been unhealthy. Alternatively, cells may have been handled too roughly during harvest or resuspension steps, or they may have been incubated too long in Buffer A. Second, it is important not to over-homogenize the cells or the nuclear pellet, as this will shear the chromatin and release DNA into the soluble fraction. DNA in the soluble fraction can interfere with subsequent purification and assay of INO80 complexes. In particular, contaminating DNA can lead to increased formation of precipitates during incubation of the extract with anti-FLAG agarose, thereby reducing the efficiency of washing and elution steps and/or may increase the abundance of contaminating DNA-binding proteins in the final purified fraction. In addition, downstream biochemical assays measure activities that are dependent on DNA, thus the potential carry-over of DNA from the extract may complicate interpretation of these assays. To avoid over-homogenization, during cell lysis it is advisable to check the percentage of trypan blue-positive cells after each stroke of the homogenizer; for HEK293 cells, near complete cell lysis typically requires 4 - 6 strokes of homogenization. It is also is important to avoid introducing air bubbles while homogenizing. The supernatant from the 120,000 x g spin in Step 4.2.8 should be a clear, non-viscous solution, with only a very minimal amount of cloudy or viscous material near the chromatin pellet or floating on top. When collecting the supernatant, one should take care not to collect any of the cloudy or viscous material, as it may include chromatin and/or DNA. Finally, it is better to avoid freezing cells before preparing nuclear extracts, since complexes prepared from frozen cells tend to exhibit lower specific activity and to include more contaminating DNA and proteins.
The optimal ratio between the amount of starting extract and anti-FLAG agarose depends on the concentration of the FLAG bait protein present in the extracts and the accessibility of the FLAG epitope and needs to be determined empirically. We typically begin immunopurification with 100 &#181;l bed volume of anti-FLAG agarose beads and 3 - 14 ml of nuclear extract; the amount of extract used depends on the goals of the experiment and availability of extract. A single step immunopurification using anti-FLAG agarose is usually sufficient to purify INO80 or INO80 subcomplexes to a degree adequate for reliable assays of their activities; however, additional purification step(s), such as gel filtration, density gradient sedimentation, or ion exchange chromatography may be needed for purification of other complexes or to remove contaminating activities that could complicate interpretation of biochemical assays.
The strategies described here should be more generally useful for studies of other chromatin remodeling enzymes as well as other large multiprotein complexes. Successful application of these strategies to the analysis of other multiprotein complexes depends on (i) identification of an individual subunit or subunit(s) that serve as scaffold(s) on which other subunits assemble and (ii) definition of specific domains or regions with which specific subsets of subunits assemble. The definition of such domains can be facilitated by analyzing the primary sequence of the core scaffold subunit(s) for evolutionarily conserved regions or regions that correspond to known structural domains.

진화 적으로 보존 SNF2 가족 염색질 리모델링 복합체는 염색질 조직 및 DNA 접근성 하나의 주요 레귤레이터이다. 이러한 개조 착체는 항상 어떤 경우에는 다양한 액세서리 단백질과 조립 및 다중 서브 유니트 매크로 분자 어셈블리를 형성하고, 중앙 SNF2 같은 ATPase의 서브 유닛을 포함한다. ATP 의존적 크로 마틴 리모델링 과정의 상세 분자를 연구하기 위해,이 착물의 활동 서브 유닛 및 / 또는 도메인 구조의 주어진 서브 세트의 기여를 이해하는 것이 중요하다. 이러한 분석은 특히 단백질 소단위 또는 도메인 구조 부족 고순도 착체 돌연변이를 생성하는 능력을 요구한다. ATP 의존적 크로 마틴 리모델링 단지의 이전 구조 기능 연구가 널리 (참고 문헌 1-4, 예를 들어, 참조)으로 인해 효모 게놈의 우수한 조작성에 효모 모델 시스템에 초점을 맞추고있다. 보존을 감안할 때orthologous 리모델링 단지 중 서브 유닛의 구성과 기능, 구조와 효모 리모델링 단지의 기능에 대한 연구는 더 높은 진핵 생물에서 그들의 대응에 중요한 통찰력을 제공하고 있습니다. 그럼에도 불구하고, 상당한 종 특이 리모델링 단지 사이에 차이가 이득이나 보존 서브 유닛의 보존 영역 내에서 종 특이 서브 유닛, 이득 또는 보존 서브 유닛의 종 - 특정 도메인의 손실 및 시퀀스 다양성의 손실 결과로 존재한다. 이러한 차이는 원칙적으로 높은 진핵 세포가 새로운 분자 세포 환경에 적응하기위한 필요에 의해 구동 될 수있다. 이뿐만 아니라 메커니즘에 대한 통찰력을 제공 할 수 있습니다 또한 ATP 의존적 크로 마틴 리모델링 과정의 기본 메커니즘을 조명하고 있지만, 때문에 따라서, 높은 진핵 리모델링 단지의 서브 유닛은 뉴 클레오 리모델링 과정에 기여하는 방법을 이해하는 것은 가치가있는 크로 마틴 구조에 의해 HIG의 유전자 발현그녀의 진핵 세포가 조절된다. 지금까지 단 생화학 적 정의 염색질 리모델링 건물 및 서브 콤플렉스를 얻기에 어려움으로 인해 일부 멀티 서브 유닛 포유 동물의 크로 마틴 리모델링 복합체의 구조 및 기능 연구를,이 제한되고있다. 우리는 부분적으로 면역 정제 안정적으로 N-말단에 FLAG 에피토프가 야생형 또는 Ino80 5-7의 돌연변이 버전 태그 발현 인간 세포에서 손상 INO80 또는 INO80 서브 콤플렉스를 제조하는데 사용되는 후술하는 절차에 이러한 어려움을 회피했다 (도 1) . 인간 세포에서 그대로 INO80 착물을 얻기 위해 중재하는 FLP 재조합 안정적 INO80 착체 8-10의 서브 유닛을 코딩 FLAG 에피토프 태그 된 cDNA를 발현하는 형질 전환 된 HEK293 세포주를 생성하는 데 사용된다. INO80 소단위의 과발현 다소 독성이있을 수 있기 때문에 분리 및 선택적 CO하에 클론 세포주를 유지하는 데 필요nditions 대규모 세포 배양의 확장에 필요한 많은 통로 중에 안정한 유전자의 발현을 확인한다. 서브 유닛의 서브 세트 만 포함 작은 INO80의 서브 콤플렉스를 얻으려면, 우리는 성공적으로이 방법을 사용했다 (그림 2A, B). 첫째, 우리는 안정적으로 특정 서브 유닛 (5)의 상호 작용에 필요한 도메인이 결여 Ino80 버전의 돌연변이를 발현하는 HEK293 FLP 인 세포주를 생성. 또한, siRNA를 매개 최저는 적절한 FLAG-태그 INO80 서브 유닛 (게시되지 않은 데이터)를 발현하는 세포에서 원하는 서브 유닛을 소모하는 데 사용됩니다. 마지막으로, 인간 INO80 착체를 정화, FLAG 아가 로스 기반 크로마토 11함으로써 효과적으로 정제 INO80 또는 INO80 서브 콤플렉스를 함유하는 최종 분획에 세포질 단백질 오염의 존재를 감소시키는, 핵 추출물에서 INO80 - 함유 분획을 풍부하게하기 위해 사용된다.

<table border="0" cellpadding="0" cellspacing="0" width="890">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Name of Reagent/Material</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:173px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:212px;">Cellgro</td>
			<td style="width:147px;">10-013-CV</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax-I (stablized glutamine)</td>
			<td>Life Technologies</td>
			<td style="width:147px;">35050-079</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>SAFC</td>
			<td style="width:147px;">12176C</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FuGENE6 transfection reagent&#160;&#160;</td>
			<td>Promega</td>
			<td style="width:147px;">E2312</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin B, sterile in PBS</td>
			<td>AG Scientific</td>
			<td style="width:147px;">H-1012-PBS</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pcDNA5/FRT vector</td>
			<td>Life Technologies</td>
			<td style="width:147px;">V6010-20</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Flp-In HEK293 cells&#160;</td>
			<td>Life Technologies</td>
			<td style="width:147px;">R780-07</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">pOG44 Flp-Recombinase Expression Vector</td>
			<td>Life Technologies</td>
			<td style="width:147px;">V600520</td>
			<td style="width:173px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">EZview&#160; Red ANTI-FLAG&#160; M2 Affinity Gel&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F2426</td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;">calf serum</td>
			<td>SAFC</td>
			<td style="width:147px;">12138C</td>
			<td style="width:173px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">TARGETplus SMARTsiRNA pool</td>
			<td>Dharmacon / Thermo Scientific</td>
			<td style="width:147px;">various</td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;">5x siRNA resuspension buffer&#160;</td>
			<td>Dharmacon / Thermo Scientific</td>
			<td style="width:147px;">#B-002000-UB-100</td>
			<td style="width:173px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine RNAiMAX&#160;</td>
			<td>Life Technologies</td>
			<td style="width:147px;">13778</td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;width:359px;">Opti-MEM Reduced Serum Medium&#160;</td>
			<td>Life Technologies</td>
			<td style="width:147px;">51985-091</td>
			<td style="width:173px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">PBS&#160;</td>
			<td style="width:212px;">Cellgro</td>
			<td style="width:147px;">45000</td>
			<td style="width:173px;">VWR</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;">TrypLE (trypsin)</td>
			<td>Life Technologies</td>
			<td style="width:147px;">12604</td>
			<td style="width:173px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1x FLAG Peptide</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F3290</td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;">Micro Bio-Spin Chromatography Column</td>
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			<td style="width:147px;">89882</td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;width:359px;">Anti-FLAG M2 antibody, mouse</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F3165</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Anti-FLAG M2 antibody, rabbit</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F7425</td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">P8340</td>
			<td style="width:173px;"></td>
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			<td>Novagen</td>
			<td style="width:147px;">70664</td>
			<td style="width:173px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:359px;"></td>
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			<td style="width:147px;"></td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;width:359px;">Equipment</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;"></td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;">Wheaton Dounce Tissue Grinders</td>
			<td>Wheaton</td>
			<td style="width:147px;">06-435C&#160;</td>
			<td style="width:173px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">JS-4.2 rotor in a J6 centrifuge&#160;</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">339080</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JA-17 rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">369691</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml polycarbonate tubes&#160;</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">355630</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 ml polycarbonate bottles&#160;</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">355655</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type 45 Ti rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">339160</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type 70.1 Ti rotor&#160;</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">342184</td>
			<td style="width:173px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">BD Clay Adams Nutator Mixer</td>
			<td style="width:212px;">BD Diagnostics</td>
			<td style="width:147px;">15172-203&#160;</td>
			<td style="width:173px;">VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glas-Col Tube/Vial Rotator</td>
			<td style="width:212px;">Glas-Col</td>
			<td style="width:147px;">099A RD4512</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR thermal cycler PTC 200</td>
			<td>MJ Research</td>
			<td style="width:147px;">PTC 200</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">roller bottle incubator</td>
			<td style="width:212px;">Bellco biotechnology</td>
			<td style="width:147px;">353348</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Immobilon-FL Transfer Membrane 7 x 8.4</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">IPFL07810</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lubricated 1.5ml microcentrifuge tubes&#160;</td>
			<td style="width:212px;">Costar</td>
			<td style="width:147px;">3207</td>
			<td style="width:173px;"></td>
		</tr>
	</tbody>
</table>

generation and purification of human ino80 chromatin remodeling complexes and subcomplexes

INO80 크로 마틴 리모델링 복합체가 ATP에 의존하는 뉴 클레오 리모델링을 촉매에 의해 뉴 클레오 역학과 DNA의 접근성을 조절한다. 인간의 INO80 단지는 Ino80, 촉매 서브 유닛과 단지의 조립을위한 발판으로 두 역할을하는 SNF2 같은 ATPase를 포함하여 14 단백질 서브 유닛으로 구성되어 있습니다. 다른 서브 유닛의 기능과 그들이 INO80 단지의 크로 마틴 리모델링 활동에 기여하는 메커니즘은 가난으로 인해 인간의 세포 또는 이종 발현 시스템에 INO80 서브 어셈블리를 생성하는 도전에 부분적으로 이해 상태로 유지됩니다. 이 조브 프로토콜은 서브 유닛 또는 서브 유닛의 일부를 부족한 사람 INO80 염색질 리모델링 서브 콤플렉스의 정화를 할 수있는 절차를 설명합니다. N-말단에 FLAG 에피토프는 Ino80 cDNA를 안정적으로 인간 배아 신장 (HEK) FLP - 중재 재조합을 사용하여 293 세포주에 도입 태그. INO80 단지의 서브 유닛의 일부는 b에있는 경우에즉 하나는 해당 서브 유닛의 조립에 필요한 플랫폼이없는 대신 Ino80 돌연변이 단백질을 발현, 삭제. 경우 개별 서브 유닛이 고갈 될 안정적 FLAG을 표현 HEK 293 세포주에이 서브 유닛을 대상으로 한 transfects s​​iRNA를가 Ino80 ATPase의 태그가. 핵 추출물을 준비하고, FLAG의 면역은 Ino80 유도체를 포함하는 단백질 분획을 풍부하게하기 위해 수행된다. 정제 INO80의 서브 콤플렉스의 조성물은 이러한 면역 블로, 실버 염색 및 질량 분석 등의 방법을 사용하여 분석 할 수있다. 이 프로토콜에 따라 생성 및 INO80 INO80 서브 콤플렉스 더욱 정돈 첨부 된 프로토콜에서 설명되는 다양한 생화학 적 분석을 이용하여 분석 될 수있다. 여기에 설명 된 방법은 모든 포유 동물의 멀티 서브 유닛 염색질 리모델링의 구조 및 기능적 특성에 대한 연구 및 수정 단지에 적용 할 수 있습니다.

Figure 1 shows a flow chart summarizing the procedures used to generate, purify, and characterize human INO80 ATP-dependent chromatin remodeling complexes.
As illustrated in Figures 2 and 3, these procedures enable the generation of both wild type INO80 and INO80 subcomplexes that lack various subunits, thereby enabling subsequent biochemical analyses of the contribution of these missing subunits to INO80&#39;s enzymatic activities. Figure 2 describes two strategies we have used to define the architecture of the INO80 complex and to generate INO80 subcomplexes. In the first, shown in Figure 2A, intact INO80 complexes are purified through FLAG-tagged versions of wild type subunits (Ies2 or INO80E). Subcomplexes can be purified through epitope-tagged versions of mutant Ino80 proteins that lack individual domains on which different sets of subunits assemble. As shown in Figure 3A, the purity of the resulting complexes is assessed using silver stained SDS-polyacrylamide gels, while the composition of the complexes is assessed using western blotting with antibodies against various INO80 subunits (Figure 3B) or by mass spectrometry (data not shown). Using this approach, we found that the subunits shown in red in Figure 2A were lost upon deletion of the Ino80 NTD (N-terminal domain); subunits shown in blue were lost upon deletion of the Ino80 HSA (Helicase SANT Associated) domain; and that the SNF2 ATPase domain, composed of SNF2N and HelicC regions (purple) separated by a long insertion region (white), was necessary and sufficient for binding to subunits shown in purple. Thus, INO80 subcomplexes (INO80&#916;N.com or INO80&#916;N&#916;HSA.com) that lack either the subunits shown in red or subunits shown in red and blue can be purified through FLAG-Ino80&#916;N or FLAG-INO80&#916;N&#916;HSA, respectively5.
It is also possible to generate subcomplexes by depleting individual subunits from cells expressing an appropriate FLAG-tagged INO80 subunit (unpublished results). In the first example shown in Figure 2B, siRNA-mediated knockdown of subunit X depletes only X from the INO80 complex, suggesting X is not required for assembly of any other subunits into INO80. In the second and third examples, siRNA knockdown of either Y or Z leads to co-depletion of both the Y and Z subunits, suggesting Y and Z assemble into the INO80 complex in a mutually dependent manner.
<img alt="Figure 1" src="/files/ftp_upload/51720/51720fig1highres.jpg" /> 
Figure 1. Flow chart describing procedures used to generate, purify and characterize human INO80 ATP-dependent chromatin remodeling complexes. F, an N-terminal in-frame FLAG epitope tag; GOI, Gene-of-interest. <a href="https://www.jove.com/files/ftp_upload/51720/51720fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/51720/51720fig2highres.jpg" /> 
Figure 2. Diagram showing the two strategies used to generate INO80 subcomplexes that contain a subset of subunits. (A) The Ino80 ATPase contains regions that function as modular scaffolds on which the other INO80 subunits assemble. (B) siRNA-mediated knockdown can be used to deplete the desired subunit (X or Y or Z) from cells expressing an appropriate FLAG-tagged INO80 subunit (e.g., FLAG-Ino80&#916;N). <a href="https://www.jove.com/files/ftp_upload/51720/51720fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/51720/51720fig3highres.jpg" /> 
Figure 3. Analysis of the composition of immunopurified INO80 complexes and subcomplexes. (A) INO80 complexes or subcomplexes were purified from cells stably expressing the indicated FLAG-tagged proteins, subjected to SDS-polyacrylamide gel electrophoresis, and visualized by silver staining. The sample in the right-most lane was prepared from parental 293 cells that express no FLAG-tagged protein; proteins that appear in this control lane are contaminants that bind non-specifically to FLAG-agarose. Labels on the left indicate bands corresponding to several INO80 subunits, and those on the right the mobilities of molecular weight markers. The position of wild type Ino80 is indicated by the black rectangle, and positions of Ino80 mutants are indicated by open circles. The lanes separated by the black line are from separate gels. (B) INO80 complexes and subcomplexes were analyzed by western blotting using antibodies against representative subunits of the NTD, HSA, and SNF2 modules. This research was originally published in The Journal of Biological Chemistry. Chen et al, Subunit Organization of the Human INO80 Chromatin Remodeling Complex. An Evolutionarily Conserved Core Complex Catalyzes ATP-dependent Nucleosome Remodeling. The Journal of Biological Chemistry Vol 286: pp. 11283-11289. &#169; 2011, by the American Society for Biochemistry and Molecular Biology. <a href="https://www.jove.com/files/ftp_upload/51720/51720fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes at JoVE.com

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

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

세대와 인간의 INO80 염색질 리모델링 단지 및 서브 콤플렉스의 정화

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

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9.8K 조회수.  Stowers Institute for Medical Research. This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography. 

비디오: Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

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 ...

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Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

인간 췌장 아일릿 절연 : 2 부 : 인간 아일 레츠의 정화와 문화

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use 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 ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

인간 CD40 - 활성화된 B 세포의 생성

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

마우스의 Cajal (ICC) 네트워크의 중간 세포의 시각화

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

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 ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA 정제에 의한 염색질 절연 (처프)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

셀 침습적 DISC1 단백질 종의 생성, 정제 및 특성

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

높은 처리량 시퀀싱을위한 고품질 염색질 면역 침전의 DNA 템플릿의 생성 (칩 SEQ)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

의 효율적인 생산 및 정제 재조합 쥐과 Kindlin-3 생물 물리학 연구 곤충 세포에서

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...