저자는 DNA 정화와 관련하여 도움이 데이터 수집 및 모니카 Pillon 동안 도움을 NSLS (브룩 헤이븐 국립 연구소)에서 PXRR 직원을 감사하고 싶습니다. 이 작품은 건강 연구의 캐나다 연구소 (걸레 67189)에 의해 지원되었다.

1. 단백질 정제 연결 유연 링커 N-(oligomerization)와 C-터미널 (DNA 바인딩) SeqA 에이즈 DNA에 1-3 회전으로 ​​구분 hemimethylated GATC 반복의 인식 도메인입니다. 본 연구의 경우, 우리는 더 oligomerization과에 의해 독점적으로 구분 직렬 GATC에게 구속력 DNA 반복을 제한 단축 링커을 방지 N-터미널 도메인의 점 돌연변이로 SeqA의 dimeric 변종 (SeqAΔ (41-59) A25R)를 사용 DNA 하나 턴 (그림 1) 2,8. <ol><li> T7 프로모터의 제어 아래에있는 플라스미드 인코딩 SeqA와 BL21 (DE3) 세포를 변환, </li><li> 판 100 μg / ML 암피실린 등의 LB-한천 플레이트의 변환 반응 </li><li> 소규모 밤 문화를 예방하기 위해 혼합 식민지를 선택 (LB 미디어를 100 μg / ML 암피실린과) </li><li> 다음날 아침은 overni의 1:100 희석을 사용하여 미디어 1 L의 예방ght 문화, </li><li> ~ 0.7의 OD 600 세포를 성장, 1 mm의 최종 농도에 이소 프로필 β-D-1-thiogalactopyranoside (IPTG)의 추가에 의해 단백질 생산을 유도, </li><li> , 궤도는 떨고으로 37 ° C에서 3 시간에 부화를 계속하고 원심 분리 (3,300그램 10 분)하여 세포를 수확 </li><li> 정화 버퍼에있는 셀 펠렛을 Resuspend하고 sonication에 의해 lyse, </li><li> 원심 분리 (39,000그램에서 40 분)에 의해 lysate를 지우고 정화 버퍼와 equilibrated 헤파린 열로 표면에 뜨는로드, </li><li> Elute SeqA는 1 M NaCl (~ 0.7 M NaCl에서 SeqA elutes)에 선형 그라디언트를 사용하여 </li><li> 함께 수영장 SeqA 함유 분수를 정화 버퍼와 equilibrated 양이온 교환 크로마토 그래피 칼럼으로 이온 샘플의 강도와 하중을 낮출 희석, </li><li> 선형 소금 그라디언트를 사용하여 순수한 SeqA은 ~ 0.4 M NaCl에서 elutes </li><li> 함께 수영장 SeqA 함유 분수를 concentrat전자 (3 MG / ML) 및 스토리지 버퍼에 저장. </li></ol> 2. DNA의 정제 <ol><li> 좋아하는 회사의 주문 보완 unmethylated과 methylated oligonucleotides, </li><li> , autoclaved ddH 2 O, 소용돌이 800 μl의 각 동결 건조 된 단일 스트랜드 DNA의 1 μmol를 해산하고 10-20 분 동안 앉아 보자 </li><li> 각 oligonucleotide에 preheated 2X 로딩 버퍼 800 μl를 추가 </li><li> 20-30 세포핵 긴 oligonucleotides를 들어, 대형 10% denaturing 젤 (160 x 250 크기 × 1 ㎜) 준비 : * 잘 10 % PAGE 믹스, 80 μl TEMED와 젤 당 800 μl 암모늄 persulfate의 80 ML을 섞어서 부어, * 일단 polymerized, 빗을 제거하고 ddH 2 O 철저으로 우물을 헹굼 * 냉각 판 등의 겔 캐스트에 젤을 조립하고 (​​1X TBE) 버퍼를 실행으로 상단과 하단 저수지를 작성, * 55 ° C에 젤을 따뜻하게 700-750 V에 젤을 미리 실행 * 실행을 중지하고 우물 thoro 씻어ughly 버퍼를 실행으로. </li><li> 90 oligonucleotides를 가열 ° C를 2 분 동안 </li><li> 소용돌이와 샘플을 스핀 즉시 젤을로드하기 전에, </li><li> ~ 700 V에서 젤을 실행하여 oligonucleotide 중간 마이그레이션되면 그만 해요. (10 % polyacrylamide 젤에서 bromophenol 파란색 ~ 60 염기 길이와 ~ 20 염기 길이 크실렌 cyanol FF oligonucleotides와 공동 마이그레이션합니다) </li><li> 젤 중지, 젤 상자에서 분해하고 스페이서를 제거 </li><li> 평평한 표면에 하나의 유리 플레이트를 제거하고 랩으로 젤을 커버 </li><li> , 젤을 돌려서 다른 유리 플레이트를 제거하고 랩으로 커버 </li><li> , DNA의 그림자를 볼 수 젤 뒤에 UV 빛과 형광 플레이트를 사용하여 밴드를 선택 </li><li> 작은 조각으로 면도날로 밴드를 잘라 멸균 15 ML 튜브에 넣어 전송 </li><li> 용출 버퍼 9 ML을 추가하고 교반으로 37 ° C에서 하룻밤 elute, </li><li> 조심스럽게 솔루션을 전송전송 아크릴 아미드의 조각을 방지하고 pH를 7 3 M의 아세트산 나트륨 (1시 10분 희석) 1 ML 플러스 차가운 100 % 에탄올 (2.5 권)의 25 ML을 추가하려면 젤 로딩 피펫 팁을 사용하여 autoclaved 원심 분리기 튜브까지, </li><li> 적어도 3 시간에 -20 ° C에서 알을 품다, </li><li> , 스핀 다운, 별도의 튜브로 표면에 뜨는 전송 </li><li> 중간 불로 속도-VAC에서 펠렛을 건조, </li><li> autoclaved ddH 2 O 400 μl의 펠렛을 Resuspend하고 신선한 튜브로 전송, </li><li> 산도 7 3 M의 아세트산 나트륨 및 100 % 에탄올 1 ML의 40 μl 추가, 잘 혼합 (소용돌이)와 실온에서 30 분 배양는 -20에서 30 분 ° C, 다음 </li><li> 18,000그램 15 분 동안 회전하고 표면에 뜨는을 취소, </li><li> 18,000그램의 6 분의 펠릿과 스핀에서 잔류 소금을 제거 70 % 감기에 에탄올 100 μl로 펠릿을 씻어. , 에탄올을 무시하고 속도 VAC에서 펠릿 건조 </li><li> autoclaved ddH 100 μl의 총 펠릿을 Resuspend <sub&gt; 2 O. , oligonucleotide의 농도를 측정 </li><li> hemimethylated DNA의 duplexes을 단련하기 위해 그들에게 물을 욕조 안에 실내 온도로 서서히 냉각 해 다음 보완 한 가닥 몰 농도를 혼합 ° C 5 분을위한 물 목욕에 95 혼합물을 가열합니다. </li></ol> 3. 단백질 DNA 복합체 형성과 분석 <ol><li> 정화 SeqAΔ의 동등한 볼륨 (41-59) - 대답 25 (81 μM)와 hemimethylated DNA (81 μM)을 혼합 </li><li> 4에 15 분 및 매장 동안 실온에서 배양 ° C 당신은을 사용 할 준비가 될 때까지, </li><li> 상업 스파 스 매트릭스 스크린을 사용하여 결정화 조건 스크린, </li><li> 초기 결정화 리드가 확인 된 후, 회절 품질 결정을 성장 조건을 최적화 </li><li> 중 25 % 최종 농도 (로 결정 솔루션에 PEG 400 현재의 양을 증가하여 발생하는 SeqA-DNA의 결정을 CryoprotectV / V) 또는 결정화 솔루션에 20 % 글리세롤 (V / V)를 추가 </li><li> 나일론 루프와 개인의 결정을 퍼 담아 액체 질소에서 그들을 플래시 동결, </li><li> 100 K. 각 크리스탈의 회절 한계를 테스트 </li></ol> 4. 대표 결과 hemimethylated GATC 서열 사이의 분리 (I), 이중의 (II) 길이하며, (iii) : hemimethylated DNA에 바인딩 SeqAΔ (41-59) A25R의 결정 구조를 얻으려면, 우리는 연속적으로 DNA에 세 매개 변수를 최적화 부재 / 5 &#39;overhangs의 존재. 전기 이동성 교대 assays는 SeqAΔ (41-59) - A25R은 우선적으로 9-10 기본 쌍 (그림 1)로 구분 GATC 반복 바인딩을 나타냅니다. 따라서, 우리는 처음에 긴 9 또는 10 bps에 의해 두 개의 hemimethylated GATC 시퀀스를 포함하는 (bps) duplexes에게 23-24 기본 쌍 검사를 실시했습니다. 세 duplexes가 잘 모양의 크리스털을 (그림 2) 굴복. 알높은 해상도 diffracted 결정 중 어느 것도, 23 9 bps의 GATC 분리가 결정에 대한 선호 것을 나타내는 나머지보다 더 X-선 diffracted 9 bps로 구분하여 두 GATC 사이트와 긴 양면을 bps하지 찬사를 보냅니다. 따라서 이후의 모든 화면 9 bps로 간 GATC 간격을 고정. 여러 DNA 분자가 결정 구에서 B-DNA 연속을 형성하기 위해 머리 - 투 - 꼬리를 쌓을 수 있기 때문에 일반적으로 DNA의 결정은 정확한 나선형 회전에 대응하는 이중 길이에 대한 선호된다. 따라서, 우리는 21 bps (즉,이 나선형 회전)에 duplexes의 전체 길이를 단축. 무딘 끝으로 21 bps 복층 아파트 회절 품질 결정 (데이터가 게재되지 않음), 크리스털를 얻을 수 않았습니다 양쪽 끝에서 염기 하나 5 &#39;오버행과 21 bps 이중 우리의 가정 원본에있는 5로 diffracted.를 얻을 수 없습니다 않았지만 회절 한계에 개선 엔드 - 투 - 엔드 이중 협회가 실제로 선호 것을 제안ING 크리스탈 포장. SeqA는 DNA의 동일한 얼굴에있는 GATC 서열과 상호 작용 때문에, DNA의 이중의 반대 얼굴은 용매에 노출되어야합니다. 더 복잡한의 결정을 개선하기 위해, 우리는 다음 이중의 반대 얼굴을 통해 크리스탈에 인접한 DNA 분자와 그루브 - 백본 상호 작용을 촉진하는 두 GATC 사이트 간의 CG의 다이 뉴클레오타이드, 방법을 포함하도록 이중의 순서를 수정 그는 지난 10 DNA의 결정을 향상하는 데 사용되었습니다. 그러나, CG - 포함 이중으로 성장 결정의 회절 제한이 CG의 다이 뉴클레오타이드 (그림 3)을 포함하지 않은 비슷한 DNA의 이중과 함께 성장 동일했다. 이 결과는 그루브 - 백본 상호 작용이 경우에 중요하지이라고 지적했다. 우리는 이후 각에 두 개의 추가 세포핵을 가진 DNA의 이중과 재배 결정을 비교하여 overhangs의 길이를 최적화5 &#39;끝. 이 변경 사항은 수정 형태에 큰 영향을 미쳤다뿐만 아니라, 회절 한계는 추가 염기가 극적으로 분자 연락처 및 강화 크리스탈 조직 (그림 3) 변경을 나타냅니다. SeqAΔ (41-59) - A25R의 결정 구조는 DNA의 이중의 자유로운 얼굴 크리스탈 주소록에 관여하지 않는 것으로 확인이 마지막 DNA의 이중에 바인딩 같은 CG-다이 뉴클레오타이드를 도입의 제한된 효과 기대. 단백질과 DNA의 상대적인 크기에도 불구하고, 대칭 친구 사이의 대부분의 상호 작용은 단백질 단백질과 단백질 DNA 상호 작용 (그림 4)에 의해 중재됩니다. 흥미롭게도,이 특정 경우에, 5 &#39;다이 뉴클레오타이드의 오버행의 유익한 효과는 의사 연속 DNA의 형성으로 인해하지 않습니다. 대신 5 &#39;거리 DNA 축에서 methylated 스트랜드 프로젝트의 끝과 왜 두 세포핵에게 w를 설명, 단지의 근위 SeqA 분자와 상호 작용<html> 오히려 엄격하게 8,11 포장 크리스탈을 변경해야합니다. <img alt="그림 1" src="/files/ftp_upload/4266/4266fig1.jpg" /> 1 그림. hemimethylated DNA에 SeqAΔ (41-59) - A25R의 바인딩. SeqA의 도메인 (A) 개략도 중재 단백질 oligomerization과 DNA 바인딩이. 기본 쌍의 증가 (을 사이에두고 두 개의 hemimethylated GATC 서열을 포함하는 DNAs과 SeqAΔ (41-59) A25R의 (B) 전기 영동 이동 분석 X). 가장 왼쪽 차선은 (공백 표시) SeqAΔ의 부재 (41-59)의 두 GATC 시퀀스 사이의 몰 5 DNAs의 혼합물, 7, 12, 21, 25, 34 기본 쌍을 포함 - A25R. (C) 다이어그램 회절 품질 결정을 얻기 위해 DNA의 duplexes에 최적화 된 세 가지 변수를 묘사. upload/4266/4266fig2.jpg &quot;강한 : 콘텐츠 폭 =&quot;6in &quot;강한 : SRC =&quot;/ files/ftp_upload/4266/4266fig2highres.jpg &quot;/&gt; 그림 2. 9 또는 10 기본 쌍을 사이에두고 두 개의 hemimethylated GATC 사이트를 포함하는 23-24 BP 긴 duplexes로 얻은 사용 oligonucleotides와 크리스탈의 상호 GATC 거리에 있습니다. 요약 다양한 효과. 결정의 모든 사진은 같은 배율로 촬영하고 규모 바는 100 μm을 나타냅니다되었습니다. 각 SeqAΔ의 해상도 제한 (41-59) A25R-DNA는 결정은 Rigaku RU-300 X-선 발생기 시스템에서 수집 된 회절 이미지를 기준으로합니다. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig2large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img alt="그림 3" src="/files/ftp_upload/4266/4266fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4266/4266fig3highres.jpg" /> 그림 3. t의 DNA를 변화의 효과가 끝납니다. 요약21 bps 긴 duplexes 9 기본 쌍을 사이에두고 두 개의 hemimethylated GATC 사이트를 포함하고 0, 1 또는 2 염기 5 &#39;최종 overhangs을 포함와 그 oligonucleotides는 사용 결정 냈다. 결정의 모든 사진은 같은 배율로 촬영하고 규모 바는 100 μm을 나타냅니다되었습니다. 각 SeqAΔ의 해상도 제한 (41-59)은-A25R-DNA 크리스탈은 (NSLS, BNL) X12C와 X29 beamlines에서 수집 회절 이미지를 기준으로합니다. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig3large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img alt="그림 4" src="/files/ftp_upload/4266/4266fig4.jpg" /> 4 그림. SeqAΔ의 맑고 포장 (41-59) - A25R는 다이 뉴클레오타이드의 오버행과 DNA에 바인딩 : (A) 상단과 측면 (B)에서 본. 비대칭 단위는 두 SeqAΔ (41-59)를-A25R-DNA의 DNA가 주황색으로 표시되는 동안 단백질은 회색으로 표시됩니다 단지가 들어 있습니다. Symmet보시지 관련 SeqAΔ (41-59) A25R-DNA 분자는 단백질과 DNA에 노란색에 흰색으로 표시됩니다. 이 수치는 PyMOL 12를 사용하여 준비되었습니다. 이 수치는 영화 일에 관련된 것은. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig4large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . 영화는 1. <a href="http://www.jove.com/files/ftp_upload/4266/4266movie1.avi" target="_blank">동영상을 보려면 여기를 클릭하십시오</a> .

<ol>
	<li>Campbell, J. L., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+oriC+and+the+dnaA+gene+promoter+are+sequestered+from+dam+methyltransferase+following+the+passage+of+the+chromosomal+replication+fork.">E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork.</a> Cell. 62, 967-979 (1990).</li><li>Guarn&#233;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+SeqA-N+filament:+implications+for+DNA+replication+and+chromosome+organization.">Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization.</a> Embo. J. 24, 1502-1511 (2005).</li><li>Brendler, T., Austin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+SeqA+protein+to+DNA+requires+interaction+between+two+or+more+complexes+bound+to+separate+hemimethylated+GATC+sequences.">Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences.</a> Embo. J. 18, 2304-2310 (1999).</li><li>Jordan, S. R., Whitcombe, T. V., Berg, J. M., Pabo, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+variation+in+DNA+length+yields+highly+ordered+repressor-operator+cocrystals.">Systematic variation in DNA length yields highly ordered repressor-operator cocrystals.</a> Science. 230, 1383-1385 (1985).</li><li>Tan, S., Hunziker, Y., Pellegrini, L., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+the+yeast+MATalpha2/MCM1/DNA+ternary+complex:+general+methods+and+principles+for+protein/DNA+cocrystallization.">Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization.</a> J. Mol. Biol. 297, 947-959 (2000).</li><li>Rice, P. A., Yang, S., Mizuuchi, K., Nash, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+IHF-DNA+complex:+a+protein-induced+DNA+U-turn.">Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn.</a> Cell. 87, 1295-1306 (1996).</li><li>Yang, W., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+site-specific+recombinase+gamma+delta+resolvase+complexed+with+a+34+bp+cleavage+site.">Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site.</a> Cell. 82, 193-207 (1995).</li><li>Chung, Y. S., Brendler, T., Austin, S., Guarne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+cooperative+binding+of+SeqA+to+a+tandem+GATC+repeat.">Structural insights into the cooperative binding of SeqA to a tandem GATC repeat.</a> Nucleic Acids Res. , (2009).</li><li>Anderson, J., Ptashne, M., Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocrystals+of+the+DNA-binding+domain+of+phage+434+repressor+and+a+synthetic+phage+434+operator.">Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator.</a> Proc. Natl. Acad. Sci. U.S.A. 81, 1307-1311 (1984).</li><li>Timsit, Y., Moras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+self-fitting:+the+double+helix+directs+the+geometry+of+its+supramolecular+assembly.">DNA self-fitting: the double helix directs the geometry of its supramolecular assembly.</a> Embo J. 13, 2737-2746 (1994).</li><li>Chung, Y. S., Guarne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+analysis+of+SeqA+bound+to+a+pair+of+hemimethylated+GATC+sites.">Crystallization and preliminary X-ray diffraction analysis of SeqA bound to a pair of hemimethylated GATC sites.</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 64, 567-571 (2008).</li><li>DeLano, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL Molecular Graphic Systems. , (2002).</li></ol>

관심 없음 충돌이 선언 없습니다.

macromolecular X-선 결정학에서 가장 큰 도전 중 하나는 회절 품질 결정을 획득하고 있습니다. 단백질 또는 단백질 DNA 단지의 경우,이 도전은 최적화되어야합니다 추가 변수로 인해 악화됩니다. 그것은 널리 DNA의 길이와 끈적 overhangs의 존재가 더 이상 가상 이중의 주요 매개 변수를 최적화하는 것입니다에 인접한 DNA 분자의 연결을 강화하기 졌다고합니다. 그러나, 우리는 이러한 overhangs의 특성과 길?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 # </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 트리스 </td><td> Bioshop </td><td> TRS003.5 </td><td></td></tr><tr><td> Ethylenediaminetetraacetic 산 (EDTA (에틸렌 다이아 민 테트라 초산)) </td><td> 피셔 과학 </td><td> E478-500 </td><td></td></tr><tr><td> Dithiotheitrol (DTT) </td><td> 바이오 기본 주식회사 </td><td> DB0058 </td><td></td></tr><tr><td> NaCl </td><td> Bioshop </td><td> SOD002.10 </td><td></td></tr><tr><td> 글리세린 </td><td> Caledon </td><td> 5350-1 </td><td></td></tr><tr><td> 자당 </td><td> 시그마 - 알드리치 </td><td> S5016-500G </td><td></td></tr><tr><td> 나트륨 dodecyl 황산 (SDS) </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td> 요소 </td><td> Bioshop </td><td> URE001.5 </td><td></td></tr><tr><td> 40% 29:1 BIS / 아크릴 아미드 </td><td> 바이오 기본 주식회사 </td><td> A0007-500ml </td><td> 4에 저장 ° C </td></tr><tr><td> 붕산 </td><td> EMD </td><td> BX0865-1 </td><td></td></tr><tr><td> 크실렌 cyanol FF </td><td> 바이오 RAD </td><td> 161-0423 </td><td></td></tr><tr><td> Bromophenol 블루 </td><td> Bioshop </td><td> BR0222 </td><td></td></tr><tr><td> 이중 조절 수직 젤 시스템 </td><td> CBC 과학 회사 주식회사 </td><td> DASG-250 </td><td></td></tr><tr><td> 인덱스 결정화 화면 </td><td> 햄튼 연구 </td><td> HR2-144 </td><td> 4에 저장 ° C </td></tr><tr><td> 마법사 I의 결정 화면 </td><td> 에메랄드 BioSystems </td><td> EBS-마법사-1 </td><td> 4에 저장 ° C </td></tr><tr><td> 마법사 II의 결정 화면 </td><td&gt; 에메랄드 BioSystems </td><td> EBS-마법사-2 </td><td> 4에 저장 ° C </td></tr><tr><td> 클래식 결정 화면 </td><td> Qiagen </td><td> 130701 </td><td> 4에 저장 ° C </td></tr><tr><td> Intelliplate 트레이 </td><td> 예술 로빈스 악기 </td><td> 102-0001-00 </td><td></td></tr><tr><td></td><td></td><td></td><td> 솔루션 단백질 정화 버퍼 : 100 MM 트리스 산도 8, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 2 MM DTT와 5 % 글리세롤. 단백질 저장 버퍼 : 20 MM 트리스 산도 8, 150 MM NaCl, 5 MM DTT, 0.5 MM EDTA (에틸렌 다이아 민 테트라 초산) 및 5 % 글리세롤. 젤 로딩 믹스 : autoclaved ddH 2 O.의 70 ML에 자당의 20g, bromophenol 파란색의 25 밀리그램, 크실렌 cyanol FF의 25 밀리그램, 10 % w / V SDS의 1 ML과 10X TBE의 10 ML을 추가 자당이 용해 될 때까지 약한 난방 저어 및 최종을 조절볼륨 100 autoclaved ddH 2 O.있는 ML 4 ° C.에 저장 2X로드 버퍼 : 젤 로딩 믹스의 10 ML에 요소의 11g을 추가합니다. 요소가 해소 될 때까지 뜨거운 접시에 젓는다. 4 2 ML 튜브 및 매장에서 나누어지는 ° C. 10X PAGE 믹스 : 믹스 요소의 420.4 g, 10X TBE의 100 ML (autoclaved), 40 % 250 ML 29:1 ddH 2 O.에서 BIS / 아크릴 아미드 완전히 용해 될 때까지 저어 및 1리터에 볼륨을 조정할 수 있습니다. 4 ° C.에서 어두운 병에 저장 10X TBE : 디졸브 108g 트리스, ddH 2 O. 1 리터에 붕산의 55g과 EDTA (에틸렌 다이아 민 테트라 초산) 9.3 g의 상온에서 압력솥 및 매장. 용출 버퍼 : ddH 2 O. 200 ML 5 M NaCl, 1 M 트리스 산도 7.5, 0.5 M EDTA (에틸렌 다이아 민 테트라 초산) 산도 8 0.4 ML 2 ML의 희석 8 ML 상온에서 압력솥 및 매장. </td></tr></tbody></tab르&gt;

iterative optimization of dna duplexes for crystallization of seqa-dna complexes

에스 케리 키아 대장균 SeqA 복제 1의 기원에서 sequestering hemimethylated GATC 클러스터의 조기 reinitiation 이벤트를 방지 DNA 복제의 부정적인 레귤레이터입니다. 원산지 외에도 SeqA은 복제 포크, 그것이 높은 명령 구조로 새로 복제 된 DNA를 구성 2 발견됩니다. SeqA 동료는 약하게 하나의 GATC 시퀀스로하지만, 여러 GATC 사이트를 포함하는 DNA의 duplexes 높은 친화 단지를 형성한다. SeqA의 최소한의 기능 및 구조 단위는이를 hemimethylated DNA 3 고친 화성 단지를 ​​형성하는 적어도 두 개의 GATC 시퀀스의 요구 사항을 설명하고, 이량 체입니다. 또한, oligomerization과 유연한 링커로 구분하여 DNA 결합 도메인 SeqA 아키텍처, 3 나선형 회전까지로 구분 GATC 반복에 바인딩 할 수 있습니다. 따라서, 분자 수준에서 SeqA의 기능을 이해하는 것은 구조적 아나가 필요합니다SeqA의 용해는 여러 GATC 시퀀스에 바인딩. 단백질 DNA의 결정에서 DNA는 단백질과 DNA의 상대적인 크기와 건축에 따라 포장의 상호 작용에 탁월한 효과 하나도 없다. 단백질은 DNA 나 발자국 DNA의 대부분을보다 큰 경우, 크리스탈 포장은 주로 단백질 단백질 상호 작용에 의해 중재됩니다. 반대로, 단백질이 DNA보다 같은 크기 또는 작은 또는 만 DNA, DNA-DNA 및 DNA-단백질 상호 작용의 일부를 포함 할 때 크리스탈 포장을 지배. 따라서, 단백질 DNA 단지의 결정은 DNA의 길이 4 DNA 종료 (둔기 나 오버행) 5-7의 체계적인 심사가 필요합니다. 이 보고서에서, 우리는 설계, 최적화, 정화 및 구조 결정에 적합한 결정을 얻을 수 SeqA의 dimeric 변형 (SeqAΔ (41-59) - A25R)으로 단지에 나란히 GATC 반복을 포함 hemimethylated의 DNA duplexes을 결정화하는 방법에 대해 설명합니다.

Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

단백질 DNA 단지의 결정 구조는 단백질 기능, 메커니즘뿐만 아니라, 특정 상호 작용의 성격에 대한 통찰력을 제공 할 수 있습니다. 여기, 우리는 함께 공동 결정의 길이, 순서 및 이중 DNA의 끝을 최적화하는 방법을보고<em&gt; 대장균</em&gt; SeqA, 복제 개시의 네거티브 레귤레이터.

SeqA-DNA 단지의 결정에 대한 DNA의 Duplexes의 최적화를 반복

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

iterative-optimization-dna-duplexes-for-crystallization-seqa-dna

Research

JoVE Journal

Biology

9.9K 조회수.  McMaster University. 단백질 DNA 단지의 결정 구조는 단백질 기능, 메커니즘뿐만 아니라, 특정 상호 작용의 성격에 대한 통찰력을 제공 할 수 있습니다. 여기, 우리는 함께 공동 결정의 길이, 순서 및 이중 DNA의 끝을 최적화하는 방법을보고 대장균 SeqA, 복제 개시의 네거티브 레귤레이터.

비디오: Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

전자 Crystallography에 의해 구조 생물학 연구에 대한 작은 막 단백질의 2 차원 결정화 평가판을 평가

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

연구

생물학

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

X - 선 Crystallography에 대한 단백질 결정화

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Lipidic Mesophases의 막 단백질의 결정화

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidic Mesophases의 막 단백질의 높은 처리량을 결정하기위한 로봇의 사용

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

식물 단백질 복합체의 번역 후 변형의 확인

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

최적화 및 활용<em&gt; 아그로 박테 리움</em에서&gt; 매개 과도 단백질 생산<em&gt;는 담배</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

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

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

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

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

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

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

비례 생체 분자 비콘을 사용하여 살아있는 세포의 단일 엔지니어링 RNA 성적 증명서의 실시간 영상

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

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

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

세대와 인간의 INO80 염색질 리모델링 단지 및 서브 콤플렉스의 정화

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