우리는 도움이 토론에 대한 Tuschl 연구소의 회원 감사합니다. MH는 도이쳐 Akademischer Austauschdienst (DAAD)가 지원됩니다. 이 작품은 스위스 국립 기금 부여 MZ에 # 3100A0 - 114001에 의해 지원되었다; TT는 HHMI의 조사이며, 자신의 실험실에서 연구는 NIH 보조금 GM073047과 MH08442과 스타 재단에 의해 지원되었다.

아래의 프로토콜 HEK293 세포가 깃발을 표현 / doxycycline와 유도시 IGF2BP1을 HA - 태그에 대한 PAR - 클립 절차를 설명합니다. 우리는 immunoprecipitation를위한 안티 - 플래그 항체를 사용합니다. PAR - 클립 immunoprecipitation를위한 효율적인 항체를 사용할 경우 모든 세포주 관심의 RNA 결합 단백질 (RBP)를 태그가 지정되지 않은 내생의 감지 수준을 표현과 함께 작동합니다. 확대 셀 <ol><li> 성장 매체 FlpIn-HEK293/TO/FLAG/HA-IGF2BP1 세포를 확장합니다. 우리는 출발점으로 100-400 X 10 6 세포 (약 10-40 15cm의 세포 배양 접시) 사이에 사용하는 것이 좋습니다. 약 80 % confluency로 성장합니다. </li><li> crosslinking 전에 14 H는 직접 세포 배양 매체 100 μm의의 최종 농도 (1 M 4 thiouridine 주식 솔루션 1:1000 V / V)에) 4 thiouridine를 추가하고 B) 국기 / HA의 표현을 유도 doxycycline (10 MG / ML doxycycline 주식 솔루션의 1:10,000 V / V) 1 μg / ML를 추가로 IGF2BP1 태그. 참고 : 대신 4 thiouridine의 당신은 또한 6 thioguanosine 100 μm의를 사용할 수 있습니다. </li></ol> UV - Crosslinking <ol><li> 판 당 10 ML 얼음처럼 차가운 PBS로 세포를 한 번 씻고 완전히 PBS를 제거합니다. </li><li> 얼음 0.15 J / cm 2의 365 nm의 Stratalinker 2400에서 자외선 (Stratagene) 또는 유사한 장치와 덮개 비추다있는 트레이에 플레이스 번호판입니다. </li><li> 50 ML의 원심 분리 튜브에 판, 전송 당 1 ML PBS에서 고무 경찰과 세포를 긁어 4 ° C에서 5 분 500 XG에서 원심 분리하여 수집하고 뜨는 폐기하십시오. 100 X 10 6 HEK293 세포 (10 15cm 접시) 약 얻을 것입니다. 서부 유럽 표준시 세포 펠렛 1 ML. </li><li> (옵션)가 세포 lysis와 직접 계속하기를 원하지 않는다면, 충격 -80에서 액체 질소와 매장의 셀입니다 펠렛 ° C.를 동결 세포 펠렛은 최소한 12 개월 저장할 수 있습니다. </li></ol> 세포 용해와 RNaseT1 다이제스트 <ol><li> 1X NP40 용해 버퍼 3 권 가교 세포의 세포 펠렛을 타고 10 분 얼음에 품어. </li><li> 4 15 분 13,000 XG에서 원심 분리에 의해 삭제 세포 lysate ° C. </li><li> 0.2 μm의 멤브레인 필터 주사기 (팔 Acrodisc 또는 이에 상응하는 금액)를 통해 필터링하여 더욱 lysate를 삭제합니다. </li><li> 22 1 U / 15 분 물 목욕에 μl와 부화 ° C.의 최종 농도 RNase T1을 (Fermentas, 10,000 U / μl) 추가 계속하기 전에 얼음 5 분 이후 쿨 반응. </li></ol> Immunoprecipitation 및 가교 대상 RNA 조각의 복구 자기 분리기를 사용하여 밖으로 건조에서 자기 구슬을 방지하기 위해 샘플 준비 전반에 걸쳐 다음 지침을 따르십시오. <ol><li> 1 2 분 동안 자기 증언대에 구슬이 들어있는 튜브를 놓습니다. </li><li> 튜브는 자기 기호에있는 동안 튜브에 버퍼를 추가합니다. </li><li> 튜브 뚜껑, 자석 구분에서 제거하고, 구슬을 resuspend. 당신은 손가락으로 튜브를 flicking하여 구슬을 resuspend 또는 5 6 세트 vortexer을 사용할 수 있습니다. </li><li> 튜브 뚜껑에 남아있을 수있는 구슬을 수집하기 위해 간단히 원심 분리기. </li><li> 필요에 따라 1-4 단계를 반복합니다. </li></ol> 자기 구슬의 준비 <ol><li> Dynabeads의 단백질 G 자성 입자의 10 μl (Invitrogen) 당 ML 세포 lysate를 전송 (이것은되어야 일반적인 실험을 위해 약. 구슬 40 50 μl) 1.5 ML의 microfuge 튜브 있습니다. 구연 산염 인산 버퍼 1 ML로 두 번 구슬을 씻으십시오. </li><li> 비드 정지의 원래 볼륨에 비해 구연 산염 인산 버퍼의 두 배 볼륨 Resuspend. </li><li> 안티 - 플래그 M2 단클론 항체의 0.25 μg (시그마) 실온에서 40 분에 회전 바퀴에서 당 ML 서스펜션과 부화를 추가합니다. </li><li> 언바운드 항체를 제거하는 구연 산염 인산 버퍼 1 ML에 두 구슬을 씻으십시오. </li><li> 비드 정지의 원래 볼륨에 비해 구연 산염 인산 버퍼의 두 볼륨에 비즈를 Resuspend. </li></ol> Immunoprecipitation (IP), 둘째 RNase T1의 소화 및 dephosphorylation <ol><li> 부분 RNase T1 취급 세포 lysate의 ML 당 신선한 항체 - 복합 자석 구슬 20 μl를 추가하고 4 1 H에 대한 회전 바퀴 15 ML의 원심 분리 튜브에 품어 ° C. </li><li> 15 50 ML 원심 분리 튜브 (Invitrogen)에 대한 자성 입자 수집기에 자기 구슬을 모아 1.5 ML의 microfuge 튜브로 전송할 수 있습니다. </li><li> IP 세척 버퍼 1 ML에 구슬에게 3 번 씻으십시오. </li><li> 100 U / μl의 최종 농도 RNaseT1을 (Fermentas, 10,000 U / μl) 추가 22 15 분 물 목욕에 비드 현탁액을 품어 ° C. 얼음 이후 쿨5 분. </li><li> 높은 소금 세척 버퍼 1 ML에 구슬에게 3 번 씻으십시오. </li><li> dephosphorylation 버퍼 1 볼륨에 Resuspend 비즈 </li><li> 0.5 U / μl의 최종 농도 송아지 장 알칼리성 인산 가수 분해 효소 (채워, 코)을 추가, 37 10 분 정학을 품어 ° C. </li><li> 인산 가수 분해 효소 세척 버퍼 1 ML 두 번 구슬 와시 </li><li> 두 폴리 뉴클레오 타이드 키나제 (PNK) 버퍼에 DTT (효소 반응에 필요한 DT​​T 농도 자기 구슬을 손상 정도로 높습니다)하지 않고 구슬을 씻으십시오. </li><li> PNK 버퍼 중 하나가 원래 비즈 볼륨 Resuspend 비즈 </li></ol> immunoprecipitated 단백질 가교 RNA 세그먼트의 Radiolabeling <ol><li> 위에서 설명한 비드 중지, 1 U / 한 원래의 구슬 볼륨 μl을 0.5 μCi / μl와 T4 PNK의 최종 농도 (코)에 Υ - 32 P - ATP를 추가합니다. 37 30 분 정학을 품어 ° C. </li><li> 100 μm의의 최종 농도를 구하고 37 또 다른 5 분 부화되지 않은 방사성 ATP를 추가 ° C. </li><li> DTT없이 PNK 버퍼 800 μl와 자성 비즈에게 5 번 씻으십시오. </li><li> SDS - PAGE 로딩 버퍼의 70 μl의 구슬을 Resuspend. </li></ol> 겔 조각에서 SDS - PAGE와 가교 RNA - 단백질 단지의 electroelution <ol><li> 95에서 열 블록에서 5 분 radiolabeled 정학을 품어 ° C 변성 및 가교 RNA와 와동과 immunoprecipitated RBP을 해제합니다. </li><li> 분리기에 자성 비즈를 제거하고 깨끗한 1.5 ML의 microfuge 관에 뜨는을 전송하기만하면됩니다. </li><li> 40 뜨는의 μl 당 우물 Novex 비스 - 트리스 4-12% (Invitrogen) 프리 캐스트의 polyacrylamide 젤을로드하고 200 55 분에 대한 겔을 실행 V. </li><li> 겔 챔버를 해체하고 한 접시에 장착 떠나, 젤 해체. phosphorimager 용지 출력에 젤의 정렬을 촉진하기 위하여, 우리는 젤의 네 모퉁이의 세에 불균형 작은 세 개의 방사성 겔 조각을 이온 주입하는 것이 좋습니다. 방사성 젤 조각는 이전에 다음 ID로 radiolabeled 합성 oligonucleotides를 정화하는 데 사용되었다 젤에서 수집한 수 있습니다. 오염을 피하기 위해 플라스틱 필름 (예 : 사란 랩)에서 젤 싸서. </li><li> 1 H에 대한 무시 phosphorimager 화면 젤 노출하고 phosphorimager에 시각화. </li><li> 오리 엔테이션에 대한 이식 겔 조각을 사용하여 phosphorimager의 인쇄물 위에 젤 맞춥니다. RBP의 예상 크기 (IGF2BP1 약. 75 KDA)에 대응하고 D - 튜브 Dialyzer 미디 튜브로 전송 800 μl 1X SDS 실행 버퍼를 추가 밴드를 잘라 버릴거야. </li><li> 2 H. 100 V에서 버퍼를 실행 1X SDS의 가교 RNA - RBP 복잡한 Electroelute 겔에서 excised 및 제조 업체의 지시에 따라 800 μl SDS 실행 버퍼에 D - 튜브 Dialyzer 미디 (Novagen)에 electroeluted. </li></ol> Proteinase K의 소화 <ol><li> 1.2 MG / ML의 최종 농도 Proteinase K (로체)의 또한 다음 electroeluate에 대하여 2X Proteinase K 버퍼의 동일한 볼륨을 추가합니다. 55 30 분 품어 ° C. </li><li> 산성 페놀 / 클로로포름 / IAA 추출 (25:24:1, 산도 4.0) 클로로포름 추출에 의해 다음에 의해 RNA를 복구합니다. 글리코겐 1 μl를 (10 MG / ML 주식) 추가 에탄올 3 볼륨을 추가하여 RNA를 침전. 10.5 μl 물에 펠렛을 디졸브. </li></ol> cDNA 라이브러리 준비와 깊은 시퀀싱 원래 작은 규제 RNAS 19 복제에 대한 설명 표준 cDNA 라이브러리 준비 프로토콜을 통해 복구된 RNA를 운반. 복구 RNA의 10.5 μl를 사용하여 20 μl 규모에서 설명하는 것처럼 첫 번째 단계, 3 &#39;어댑터 내고이 진행되었습니다. Solexa 순서 어댑터 설명 집합을 사용합니다. 복구 RNA의 양에 따라 삽입하지 않고 5&#39; - 어댑터 - 3&#39; - 어댑터 제품은 추가 PCR 밴드로 cDNA의 증폭 후 검색됩니다. 이러한 경우에는, 소비세 3 % NuSieve에서 예상 크기의 긴 PCR 제품은 낮은 용융 점 아가로 오스 겔, Solexa 기술을 사용하여 GelElute 키트 (Qiagen)와 순서를 사용하여 겔 조각에서 PCR 제품을 elute. 한 Solexa 순서 실행은 보통 6 천만 순서 사이 내실수 RNA 바인딩 단백질의 바인딩 사이트의 transcriptome 넓은 범위에 대한 충분한 것을 읽습니다. Bioinformatic 분석 순서주의 bioinformatic 분석 순서 대 번역되지 않은 코딩, 요구 사항 등 RNA 인식 요소 RBP이 가진 기본 바인딩 지역 (exonic 대 intronic으로 심사 RBP에 대한 RNA 구속력이 사이트에 의미있는 통찰력을 얻기 위해해야​​ 할 일이 읽습니다 순서). 읽는 순서는 게놈과 동부 표준시 데이터베이스에 대해 정렬해야합니다. 우리는 일반적으로 읽습니다 mappi를 사용하여을 한 불일치, 삽입 또는 시퀀스 클러스터 구축을 삭제까지 고유 게놈에 NG가 나서 더 이상 분석 수 읽습니다. 클러스터 합성의 특성 변이의 빈도는 6 - SG를 사용할 때 전환 4 - SU와 G를 사용하는 경우, C 전환하기 위해 T를 읽는 성공적으로 가교 시퀀스 나타내는 있습니다. 우리의 경험에서 4 - SU으로 표시 uncrosslinked RNAS는 약 20 %의 배경 돌연변이 속도를 보여줍니다. 이 속도는 약으로 증가합니다. crosslinking시 50~80%. bioinformatic 분석에 대한 자세한 설명은 Hafner 외 의해 출판의 보충 자료에서 찾을 수 있습니다. 18 선택 단계 총 RNA에 4 thiouridine의 정관 수준의 결정 안정 100 μm의 사전 수확 4SU 16 H와 보충 매체 성장 후 관심 RBP을 표현하는 세포 라인에서 총 RNA를 분리. 제어로, 수확 전지는 4SU 또한없는 성장. 제조 업체의 지침에 따라 세탁 세포 펠렛을 Trizol 시약 (시그마) 3 권 외에하여 총 RNA를 분리. 또한 제조 업체의 프로토콜에 따라 Qiagen RNeasy를 사용하여 총 RNA를 정화했다. RNA 분리 및 분석하는 동안 4SU의 산화를 방지하기 위해 세척 버퍼와 후속 효소 단계를 0.1 MM dithiothreitol (DTT)을 추가합니다. 다이제스트 및 HPLC 분석을 위해 하나의 nucleosides에 dephosphorylated 총 RNA는 20 전에 설명했다. 간단히, 30 μl 볼륨, 37 16 H에 대한 전체 RNA 투석을 40 μg을했다 품어 ° 0.4 U 세균 알칼리성 인산 가수 분해 효소 (생물 워싱턴)와 0.09 U의 뱀 독이와 C phosphodiesterase (워싱턴 생물). 참조 표준으로, 합성 4SU - 라벨 RNA (우리가 standardly CGUACGCGGAAUACUUCGA (4SU) U 사용) 또한 효소 소화를 완료하는 데 그것을 제목을 사용합니다. Supelco 디스커버리 C18 (보세 위상 실리카 5 μm의 입자, 250 X 4.6 mm) 반대 위상 열 (펠폰트 PA, 미국)의 HPLC에 의해 ribonucleosides의 결과 혼합물을 분리합니다. HPLC 버퍼는 3 % acetonitrile (A)와 물에 90 % acetonitrile (B)에 0.1 M TEAA 있습니다. 15 분, 20 분 0으로 10 % B, 30 분 10-100%의 B에 대한 0 % B : isocratic 그라데이션을 사용합니다. 사이에 적용 세척은 HPLC의 컬럼을 청소 실행 5 분 100 % B을 적용합니다. 대표 결과 <img src="/files/ftp_upload/2034/2034fig1.jpg" alt="그림 1" /> 그림 1 (오른쪽 패널)은 세포 라인 플래그를 표현 / 4 - SU 6 - SG와 함께 IGF2BP1 HA - 태그와 수행 파 클립의 대표적인 결과를 보여줍니다. IGF2BP1 6 - SG의 crosslinking 효율 4 - SU에 대한 crosslinking 효율보다 낮은합니다. 낮은 crosslinking 효율 풍부한 세포 RN​​AS의 조각에서 파생된 시퀀스의 높은 배경에 결과되며, 따라서 덜 효율적인 photoreactive nucleosides를 사용할 때 실험을 축소 고려해야합니다. 그림 1의 왼쪽 패널은 잠재적으로 기존의 UV 254 나노미터 crosslinking에 비해 PAR - 클립에 사용할 수있는 다른 photoreactive 유리딘의 analogs를 사용하여 비교를 보여줍니다. 올바른 길이의 방사능 밴드의 강도는 당신에게 작은 RNA 순서 프로토콜 (소형의 cDNA 라이브러리 준비에 대한 단계별 설명을 통해 파 클립 실험이 효과가있다 당신이 수행하는 충분한 RNA을 고립 여부 좋은 생각을 제공합니다 RNAS 배열은) 19에서 찾을 수 있습니다. 6 - SG를 사용할 때 전환 4 - SU와 G를 사용하여 C 전환하는 합성 읽는, T의 특성 변이의 빈도는 성공적으로 가교 시퀀스 나타내는 있습니다. 우리의 경험에서 4 - SU으로 표시 uncrosslinked RNAS는 약 20 %의 배경 돌연변이 속도를 보여줍니다. 이 속도는 약으로 증가합니다. crosslinking시 50~80%.

<ol>
	<li>Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+regulons:+coordination+of+post-transcriptional+events.">RNA regulons: coordination of post-transcriptional events.</a> Nat. Rev. Genet. 8 (7), 533-533 (2007).</li><li>Tenenbaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+mRNA+subsets+in+messenger+ribonucleoprotein+complexes+by+using+cDNA+arrays.">Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.</a> Proc. Nat. Acad. Sci. 97 (26), 14085-14085 (2000).</li><li>Gerber, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+mRNAs+associated+with+the+translational+regulator+PUMILIO+in+Drosophila+melanogaster.">Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster.</a> Proc. Nat. Acad. Sci. 103 (12), 4487-4487 (2006).</li><li>Lopez de Silanes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+target+RNA+motif+for+RNA-binding+protein+HuR.">Identification of a target RNA motif for RNA-binding protein HuR.</a> Proc. Nat. Acad. Sci. 101 (9), 2987-2987 (2004).</li><li>Greenberg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+light-induced+crosslinking+of+mRNA+to+proteins.">Ultraviolet light-induced crosslinking of mRNA to proteins.</a> Nucl. Acids Res. 6 (2), 715-715 (1979).</li><li>Wagenmakers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+of+mRNA+to+proteins+by+irradiation+of+intact+cells+with+ultraviolet+light.">Cross-linking of mRNA to proteins by irradiation of intact cells with ultraviolet light.</a> Eur. J. Biochem. 112 (2), 323-323 (1980).</li><li>Mayrand, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+nuclear+ribonucleoprotein:+identification+of+proteins+in+contact+with+poly(A)++heterogeneous+nuclear+RNA+in+living+HeLa+cells.">Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.</a> The Journal of Cell Biology. 90 (2), 380-380 (1981).</li><li>Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+heterogeneous+nuclear+RNA-protein+complexes+in+vivo+with+monoclonal+antibodies.">Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.</a> Mol. Cell. Biol. 4 (6), 1104-11 (1984).</li><li>Adam, S. A., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+proteins+associated+with+mRNA+and+hnRNA+in+infected+HeLa+cells.">Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells.</a> J. Virol. 61 (10), 3276-3276 (1987).</li><li>Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP+identifies+Nova-regulated+RNA+networks+in+the+brain.">CLIP identifies Nova-regulated RNA networks in the brain.</a> Science. 302 (5648), 1212-1212 (2003).</li><li>Licatalosi, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HITS-CLIP+yields+genome-wide+insights+into+brain+alternative+RNA+processing.">HITS-CLIP yields genome-wide insights into brain alternative RNA processing.</a> Nature. 456 (7221), 464-464 (2008).</li><li>Yeo, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+code+for+the+FOX2+splicing+regulator+revealed+by+mapping+RNA-protein+interactions+in+stem+cells.">An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells.</a> Nat. Struct. Mol. Biol. 16 (2), 130-130 (2009).</li><li>Sanford, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+factor+SFRS1+recognizes+a+functionally+diverse+landscape+of+RNA+transcripts.">Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.</a> Genome Res. 19 (3), 381-381 (2009).</li><li>Granneman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+binding+sites+on+U3+snoRNA+and+pre-rRNA+by+UV+cross-linking+and+high-throughput+analysis+of+cDNAs.">Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs.</a> Proc. Nat. Acad. Sci. , (2009).</li><li>Guil, S., Caceres, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifunctional+RNA-binding+protein+hnRNP+A1+is+required+for+processing+of+miR-18a.">The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a.</a> Nat. Struct. Mol. Biol. 14 (7), 591-591 (2007).</li><li>Chi, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Argonaute+HITS-CLIP+decodes+microRNA-mRNA+interaction+maps.">Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.</a> Nature. 460 (7254), 479-479 (2009).</li><li>Zisoulis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+discovery+of+endogenous+Argonaute+binding+sites+in+Caenorhabditis+elegans.">Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans.</a> Nat. Struct. Mol. Biol. , (2010).</li><li>Hafner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+identification+of+RNA-binding+protein+and+microRNA+target+sites+by+PAR-CLIP.">Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.</a> Cell. , (2010).</li><li>Hafner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+microRNAs+and+other+small+regulatory+RNAs+using+cDNA+library+sequencing.">Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing.</a> Methods. 44 (1), 3-3 (2008).</li><li>Andrus, A., Kuimelis, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+composition+analysis+of+nucleosides+using+HPLC.">Base composition analysis of nucleosides using HPLC.</a> Current Protocols in Nucleic Acid Chemistry. Chapter 10 (Unit 10.6), (2001).</li></ol>

TT는 Alnylam 제약에 cofounder 과학 고문과 레굴루스 치료제에 대한 고문이다.

버퍼 및 시약 성장 매체 HEK293 세포 <ul><li> DMEM </li><li> 10% FBS </li><li> 이 MM L - 글루타민 </li><li> 100 U / ML 페니실린 </li><li> 100 U / ML 스트렙토 마이신 </li><li> 100 μg / ML hygromycin </li><li> 15 μg / ML blasticidin </li></ul> 4 thiouridine 주식 솔루션 (1 M) <ul><li> 260.27 MG 4 thiouridine </li><li> 1 ML DMSO </li></ul> Doxycyclin 주식 (10 MG / ML) <ul><li> 10 MG doxycyclin </li><li> 1 ML DMSO </li></ul> 1X NP40의 용해 버퍼 DTT와 단백 분해 효소 억제제없이 5 배 버퍼의 재고를 준비합니다. 직접 실험을하기 전에 DTT와 프로 테아제 억제제를 추가합니다. <ul><li> 50 MM HEPES, pH를 7.5 </li><li> 150 MM KCl </li><li> 이 MM EDTA (에틸렌 다이아 민 테트라 초산) </li><li> 1 ㎜ NaF </li><li> 0.5 % (V / V) NP40 </li><li> 0.5 MM DTT </li><li> 전체 EDTA (에틸렌 다이아 민 테트라 초산) - 무료 프로 테아제 억제제 칵테일 (로체) </li></ul> 구연 산염 인산 버퍼 <ul><li> 4.7 g / L 구연산 산성 </li><li> 9.2 g / L 나 2 HPO 4 </li><li> 산도 5.0 </li></ul> IP - 세척 버퍼 <ul><li> 50 MM HEPES - 코, 산도 7.5 </li><li> 300 MM KCl </li><li> 0.05 % (V / V) NP40 </li><li> 0.5 MM DTT (실험 바로 앞에 추가) </li><li> 전체 EDTA (에틸렌 다이아 민 테트라 초산) - 무료 프로 테아제 억제제 칵테일 (로체) (직접 실험을하기 전에 추가) </li></ul> 높은 소금 세척 버퍼 <ul><li> 50 MM HEPES - 코, 산도 7.5 </li><li> 500 MM KCl </li><li> 0.05 % (V / V) NP40 </li><li> 0.5 MM DTT (실험 바로 앞에 추가) </li><li> 전체 EDTA (에틸렌 다이아 민 테트라 초산) - 무료 프로 테아제 억제제 칵테일 (로체) (실험 바로 앞에 추가) </li></ul> Dephosphorylation 버퍼 <ul><li> 50 MM 트리스 - HCL, pH를 7.9 </li><li> 100 MM NaCl </li><li> 10 MM MgCl 2 </li><li> 1 ㎜ DTT </li></ul> 인산 가수 분해 효소 세척 버퍼 <ul><li> 50 MM 트리스 - HCL, pH를 7.5 </li><li> 20 MM EGTA </li><li> 0.5 % (V / V) NP40 </li></ul> DTT없이 폴리 뉴클레오 타이드의 키나제 (PNK) 버퍼 <ul><li> 50 MM 트리스 - HCL, pH를 7.5 </li><li> 50 MM NaCl </li><li> 10 MM MgCl 2 </li></ul> PNK 버퍼 <ul><li> 50 MM 트리스 - HCL, pH를 7.5 </li><li> 50 MM NaCl </li><li> 10 MM MgCl 2 </li><li> 5 MM DTT </li></ul> SDS 페이지 로딩 버퍼 <ul><li> 10 % 글리세롤 (V / V) </li><li> 50 MM 트리스 - HCL, pH를 6.8 </li><li> 이 MM EDTA (에틸렌 다이아 민 테트라 초산) </li><li> 2퍼센트 SDS (W / V) </li><li> 100 MM DTT </li><li> 0.1 % bromophenol 블루 </li></ul> 2X Proteinase K 버퍼 <ul><li> 100 MM 트리스 - HCL, pH를 7.5 </li><li> 150 MM NaCl </li><li> 12.5 MM EDTA (에틸렌 다이아 민 테트라 초산) </li><li> 2퍼센트 (W / V) SDS </li></ul>

par-clip - a method to identify transcriptome-wide the binding sites of rna binding proteins

RNA의 성적은 RNA 결합 단백질의 수백 (RBPs)와 microRNA 함유 자주 dependently 셀 타입에 표현 ribonucleoprotein의 단지 (miRNPs를.)와 상호 작용하여 포스트 transcriptional 유전자 규정의 대상이 아르 이해하는 방법을 다음과 RNA 바인딩 요소의 상호 작용은 개인 성적, 생체내 단백질 - RNA 상호 작용 1 필요의 고해상도지도의 규정에 영향을 미칩니다. 유전자, 생화학 및 전산 접근 방식의 조합은 일반적으로 RNA - RBP 또는 RNA - RNP 상호 작용을 파악하기 위해 적용됩니다. immunopurified의 RBPs과 관련된 RNAS의 Microarray 프로 파일링 (RIP - 칩) 2 transcriptome 수준 목표를 정의하지만, 그 응용 프로그램은 kinetically 안정적인 상호 작용에만 드문 경우 3,4는 RBP 인식 요소 (RRE을 식별할 수의 특성으로 제한됩니다 ) 긴 목표 RNA 이내. 더 직접적인 RBP 대상 사이트 정보 가교 RNA 세그먼트와 cDNA 시퀀싱 (CLIP) 10의 절연 다음 immunoprecipitation 7-9로 생체내의 자외선 crosslinking 5,6에 결합하여 얻을 수 있습니다. 클립 RBPs 11-17의 다수의 대상을 식별하는 데 사용되었다. 그러나, 클립 UV 254 nm의 RNA - 단백질 crosslinking의 낮은 효율에 의해 제한되며, crosslink의 위치는 어려운 배경 이외의 가교에서 UV - 가교 대상 RNA 세그먼트를 분리하고, 합성 가교 조각 이내에 쉽게 식별이되지 않습니다 RNA는 또한 샘플에 존재 조각. 우리는 높은 해상도와 셀룰러 RBPs 및 miRNPs의 transcriptome 전체 바인딩 사이트에서 확인하기 위해 강력한 세포 기반 crosslinking 접근을 개발 우리가 용어 PAR - 클립 (Photoactivatable - Ribonucleoside 강화 Crosslinking 및 Immunoprecipitation) (개요에 대한 그림. 1A를 참조하십시오 방법). 방법은 살아있는 세포에 의해 초기 RNA의 성적에 같은 4 - thiouridine (4 - SU)과 6 - thioguanosine (6 - SG)로 photoreactive ribonucleoside의 analogs의 결합에 의존하고 있습니다. 365 NM의 자외선에 의한 세포의 방사선이 상호 작용 RBPs에 photoreactive 뉴클레오 - 라벨 세포 RN​​AS의 효율적 crosslinking을 유도. 관심 RBP의 Immunoprecipitation은 가교와 coimmunoprecipitated RNA의 분리옵니다. 절연 RNA는 cDNA 라이브러리로 변환하고 깊은 Solexa 기술을 사용하여 합성 수 있습니다. PAR - 클립 준비한 cDNA 라이브러리 중 하나 특징은 crosslinking의 정확한 위치가 합성 cDNA에 거주하는 돌연변이에 의해 식별 될 수있다는 것이다. 변이를 아데노신에 구아 노신 6 - SG 결과를 사용하는 반면 cytidine 전환 4 - SU을 사용하여, 가교 시퀀스 thymidine. 가교 시퀀스에있는 돌연변이의 존재는 그것이 가능한 풍부한 휴대 RNAS에서 파생 시퀀스의 배경에서 분리 수 있습니다. 다양한 RNA 바인딩 단백질의 숫자 방법의 응용은 Hafner 외에 보도되었다. 18

Watch this Scientific Journal Video about PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins at JoVE.com

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA의 성적표는 횡단 행동 RNA 결합 단백질 (RBPs)의 다수에 의해 중재됩니다 광범위한 posttranscriptional 규정이 적용됩니다. 여기 우리는 정확하고 transcriptome 전체 규모 RBPs의 RNA 결합 사이트를 식별할 수있는 generalizable 방법을 제시.

PAR - 클립 - RNA 단백질의 바인딩 Transcriptome 전체 바인딩 사이트를 식별하는 방법

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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Biology

53.2K 조회수.  Rockefeller University. RNA의 성적표는 횡단 행동 RNA 결합 단백질 (RBPs)의 다수에 의해 중재됩니다 광범위한 posttranscriptional 규정이 적용됩니다. 여기 우리는 정확하고 transcriptome 전체 규모 RBPs의 RNA 결합 사이트를 식별할 수있는 generalizable 방법을 제시.

비디오: PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

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생물학

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

P2X2 리간드 - 게이 티드 양이온 채널과 상호 작용하는 단백질을 확인하는 Proteomics

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Methyltransferases의 프로 파일링 및 기타 S - adenosyl - L</sub캡처 컴파 운드 질량 분광법 (CCMS)에서> - 호모 시스테인 결합 단백질

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

바인딩 메커니즘을 결정하기 위해 가변 농도 등온 적정의 열량의 데이터 세트를 수집

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 개인 염기 해상도와 단백질 RNA 상호 작용의 Transcriptome 전체 매핑

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

풀다운 Calmodulin 결합 단백질의

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

성장 팩터 Progranulin와 상호 작용하는 단백질을 식별하기 위해 수정된 효모 - 2 하이브리드 시스템

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

미세 규모 Thermophoresis에 의한 결합력 결정 단백질 정제 - 무료 방법

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Electrochemiluminescence의 면역에 의한 혈청 심장 마이 오신 결합 단백질-C의 결정에 대한 민감하고 구체적인 정량 방법

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...