We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1. 해물 준비 <ol><li> P. 성장 원하는 OD에 LB에 루기 노사 세포. 참고 : 지침 : 로그 상 문화에 대한 고정 위상 문화에 대한 ≈ 100 ml의 문화 / 샘플 ≈ 500㎖의이 배양 / 샘플 (외경 600 ㎚ = 0.5)를 사용합니다. </li><li> 5,000 x g에서 20 분 동안 원심 분리하여 펠렛. </li><li> 재현 탁 0.5 ~ 1 ml의 용해 버퍼에 펠렛의 g (6.7 MM의 MES, 6.7 mM의 HEPES, 200 mM의 NaCl을, 6.7 mM의 한국 공항 공사, DDT 1 ㎜, pH를 7.5)와 단백질 분해 효소 억제제 (완전 미니, EDTA 무료)를 추가 할뿐만 아니라 DNaseI있다. </li><li> 를 Lyse 20,000 psi에서 프랑스의 압력 셀을 통해 3 통로에 의해 세포 (자료 목록 참조). </li><li> 초 원심 분리기 4 ° C에서 1 시간 동안 100,000 x g에서 세포 용 해물. </li><li> 뜨는을 저장 (2 단계로 이동합니다). </li><li> 피펫 팅 아래에 의해 용해 버퍼 1 배 1 mL를 펠렛을 씻으십시오. </li><li> 초 원심 분리기 4 ℃에서 1 시간 동안 100,000 x g에서. </li><li> 에 펠렛을 플래시 동결막 단백질 (단계 3 참조)의 포착을 위해 사용될 때까지 -20 ℃에서 액체 질소 저장. </li></ol> C 디 GMP 무료 및 기타 뉴클레오티드 2. 제거 (가용 분 만 해당) <ol><li> 차가운 용해 완충액 10 mL로 PD10 탈염 컬럼 (참조 자료 목록)를 씻으십시오. </li><li> 뉴클레오티드를 제거하기 위해 상등액 PD10 상 (≈ 1 ml)에 붓는다. </li><li> 4 ml의 차가운 용해 완충 용액 (500 μL 단계)로 용출시켰다. </li><li> 브래드 포드 분석에 의해 결정되는 곳 분수를 선택하고 그들을 풀. </li></ol> 3. 펠렛의 재 부상과 가용화 (막 분수 전용) <ol><li> (세제)없이 1X 캡쳐 버퍼 500 μl에 1,000에 펠렛을 재현 탁 (표 1 참조). 참고 : 펠릿 재현 탁하기 어렵다. 대략 잘 솔루션을 균질화 주사기 (27) G를 사용 후, 펠렛을 재현 탁에 그것은 피펫 먼저 피펫 최대 및 아래 좋습니다. </li><li> 1 % 추가 (W / V) N 도데 실-β-D-maltopyranoside (DDM). </li><li> 회전 바퀴에 적어도 2 시간 (또는 O / N) 4 ° C에서 품어. </li><li> 초 원심 분리기 4 ℃에서 1 시간 동안 100,000 x g에서. </li><li> 뜨는을 수확. </li></ol> 4. 단백질 농도 측정 <ol><li> (막 분획에 대한 분석으로 BCA) 브래드 포드 분석으로 단백질 농도를 측정한다. </li><li> 10 ㎎ / ㎖에 총 단백질 농도를 설정합니다. </li></ol> 5. 캡처 <ol><li> GDP, GTP, ATP, CTP의 1 ㎜, 5 배의 캡처 버퍼 (100 mM의 HEPES, 250 mM의 한국 공항 공사, 50 mM의 MgAc, 5 % 글리세롤, 산도 7.5) (표 1) 20 μL와 단백질 300 μg의 혼합. H 2 O. 100 μL에 대한 반응 볼륨을 조절 주 : 전량 경쟁 제어시 CDG 및 C-CC 디 GMP의 볼륨을 포함한다 (단계 5.3 및 표 2 참조). 참고 : 모든 실험은 200에서 수행되었다# 181; L (12) 튜브 PCR 스트립 (온도 과학). 참고 : 막 분획을 위해, 항상 임계 미셀 농도 (8) M 우레아의 단백질 가용화 단계까지 (7.1 단계) (V / W 0.01 %) 이상 DDM 농도를 유지해야합니다. </li><li> 회전 바퀴에 30 분 동안 4 ° C에서 품어. </li><li> 10 μM C​​DG-CC (최종 농도)를 추가합니다. 주 : ( &quot;비드 제어&quot;로 칭함) CDG-CC없이 제어하고, 1 mM의 C 디 GMP ( &quot;대회 관리&quot;)가 보충 된 제어를 포함 (표 2 참조). 주 : CDG-CC 농도는 1 내지 10 μM로 조절 될 수있다. </li><li> 어둠 속에서, 회전 바퀴에 적어도 2 시간 (O / N의 막 분획) 4 ° C에서 품어. </li><li> CaproBox를 사용하여 4 분 동안 자외선과 CDG-CC의 반응 부분의 활성화에 의해 짧은 스핀, 크로스 링크, 후 (소스 = 310 nm의, 조도 ≥10 mW의 / cm² 이상, 거리 재료 목록을 참조 λ = 2cm). 주 : 스트립 가교 결합 전에 뚜껑을 제거합니다. </li><li> 부드럽게 균질화, 25 ㎕의 5 배 세척 버퍼 (5 M의 NaCl 250 mM 트리스, 산도 7.5) 잘 재 부유 스트렙 타비 딘 자석 구슬의 50 μl를 추가합니다. </li><li> 회전 바퀴에 1 시간 동안 4 ° C에서 품어. </li></ol> 6. 세척 단계 참고 : (자석 : 재료 목록 참조). 자석 PCR 스트립 뚜껑에 자석 구슬의 캡처, 시작합니다. 그 다음 세척 용액을 포함하여 새로운 PCR 스트립을 대체. 자석을 제거하고 구슬을 재현 탁하고, 2 분을 품어. 스핀 다운과 신선한 뚜껑으로 뚜껑을 교체합니다. <ol><li> 세척 단계 (가용 분획 만) <ol><li> 200 μL 1X 세척 버퍼에 6 회 반복한다. </li><li> 200 μL를 HPLC 등급 H 2 O에 한 번 세척 </li><li> 200 μL 80 % 아세토 니트릴 6 회 반복한다. </li><li> 200 μL를 HPLC 등급 H 2 O 2 회 반복한다 </li></ol></li><li> 세척 단계 (membra북동 부분 만) <ol><li> + 0.1 % DDM 200 μL 1X 세척 버퍼에 5 회 반복한다. </li><li> + 0.05 % DDM 200 μL 1X 세척 버퍼하는 과정을 2 회 반복한다. </li><li> + 0.025 %의 DDM 200 μL 1X 세척 버퍼에 한 번 씻으십시오. </li><li> + 0.0125 %의 DDM 200 μL 1X 세척 버퍼에 한 번 씻으십시오. </li><li> 200 ㎕의 100 mM의 ABC에서 3 회 반복한다 (탄산 수소 암모늄, NH 4 CO 3) + 2 M 우레아. </li></ol></li></ol> 7. MS 샘플 준비 <ol><li> 20 μl의 100 mM의 ABC (막 분획에 대한 + 8 M 우레아 100 mM의 ABC)에 (직접 뚜껑) 구슬을 재현 탁 1.5 ml의 튜브에 전송합니다. </li><li> 막 분획은 500 rpm으로 흔들면서 5 분 동안 60 ℃에서 배양한다. </li><li> 200 mM의 TCEP (트리스 (2- 카르복시 에틸) 포스 핀) 0.5 μL를 첨가하고, 500 rpm으로 흔들면서, 60 ℃에서 1 시간 동안 배양한다. 25 ℃로 냉각시킨다. </li><li> 를 떨고, 요오도 아세트 아미드 새로 제조 된 400 mm의 0.5 μl를 추가하고 30 분 동안 25 ° C에서 배양500 rpm으로 어둠 속을 t. </li><li> 0.5 ㎕의 0.5 M N의 아세틸 시스테인을 추가하고 500 rpm으로 진탕, 10 분 동안 25 ° C에서 품어. </li><li> 막 분획 만 : 1 μL리스-C를 추가하고 37 ° C, O / N 품어. </li><li> 2 μg의 트립신을 추가하고 500 RPM (건조를 방지하기 위해 파라 필름에 포장) 진탕, 37 ° C에서 O / N을 배양한다. 주 : 샘플이 단계에서 -20 ℃에서 저장 될 수있다. 참고 : 막 분수는 : 트립신을 첨가하기 전에 &lt;2 M의 요소 농도를 조정하는 100 mM의 ABC를 추가합니다. </li><li> 간단히 튜브를 스핀 다운 자석 구슬을 수집합니다. </li><li> 새로운 1.5 ML 튜브에 뜨는을 전송 (구슬이 남아있는 경우이 단계를 반복). </li><li> 5 μL 5 % TFA (트리 플루오로 아세트산) + 1 μL 2 M 염산 (막 분획 15 ㎕의 5 % TFA + 5 μL 2 M 염산)를 추가합니다. </li><li> 조건 C18를 MicroSpin 열 μL 아세토 니트릴 150 (둥지 그룹, MA, USA) (스핀 2,400 rpm에서 20 초). </li><li> 동등0.1 % TFA (스핀 20 초, 2,400 RPM) μL C18 컬럼 (150)와 2 번 librate. </li><li> 샘플을 넣고 2 분, 2,000 rpm으로 스핀. </li><li> 흐름을 통해 열 상을 다시 넣고 회전 단계 (2 분, 2,000 RPM)를 반복합니다. </li><li> 0.1 % TFA μL (150)로 3 회 세척, 5 % 아세토 니트릴 (20 초, 2,400 RPM). </li><li> 새로운 튜브를 취하여 150 μL, 0.1 % TFA 회 용리, 50 % 아세토 니트릴 (2 분, 2,000 RPM). </li><li> 속도 진공 청소기의 펩티드를 건조. </li><li> 40 μL의 98 % H 2 O 2 % 아세토 니트릴, 0.15 % 포름산에서 재현 탁. </li><li> 초음파 처리 20 초 (펄스주기 0.5, 진폭 100 %; 재료 목록 참조), 5 초, 12,000 RPM (벤치 탑 원심 분리기)를 스핀 다운. 소용돌이 10 초, 5 초, 12,000 rpm으로 스핀 다운. LC-MS / MS 분석을위한 HPLC 바이알로 옮긴다. </li><li> -20 ° C에서 동결. 주 : 샘플이 단계에서 -20 ℃에서 저장 될 수있다. </li></ol> 8. LC-MS / MS 분석 <ol><li> 나노 LC를 실행 (나노 LC 시스템) equippRP-HPLC 컬럼 (75 μm의 X 37cm) C18 수지 충전과 ED (매직 C18 AQ이 3 μm) 95 % 용매 A (0.15 % 포름산, 2 % 아세토 니트릴), 5 % 용매 B의 선형 구배를 사용하여 ( 0.2 μL / 분의 유속으로 60 분에 걸쳐 용매 B 35 % 98 % 아세토 니트릴, 0.15 % 포름산). </li><li> (전기 분무 이온 원에 연결된 이중 - 압력 LTQ Orbitrap Velos 질량 분광계) LC-MS / MS를 이용하여 펩티드를 분석한다. 주 : 데이터 수집 모드는 MS가 (20)에서 가장 강한 이온의 선형 이온 트랩에 MS / MS 스캔이어서 60,000 FWHM의 해상도 질량 분석기의 FT 부 스캔 한 고해상도를 수득 하였다. MS / MS 시도의 효율을 높이기 위해 충전 상태 스크리닝 잠정은 미사용 배제하는 것을 가능하게하고, 단독 이온을 충전 하였다. 전구체 100 이온 수를 초과 할 때 담합에 의한 분해가 트리거했다. 동적 제외 기간은 30 초로 설정 하였다. 이온 축적 시간은 300 밀리 초 (MS) 및 (50)에 설정된밀리 초 (MS / MS). </li></ol> 9. 데이터베이스 검색 <ol><li> P. 다운로드 aeruginosa의 NCBI 데이터베이스 NCBI 홈페이지를 통해 ( <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ). </li><li> MassMatrix 변환 도구 (사용 마스코트 일반 파일에 MS 원시 스펙트럼 (MGF) 변환 <a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py을</a> ). </li><li> P.에 대해 마스코트 버전 2.3을 사용하여이 MGF 파일을 검색 앞으로 함유 역 미끼 단백질 항목 aeruginosa의 NCBI 데이터베이스. </li><li> 두 개의 완전히 트립신 펩티드의 분열을 놓친 용인 (프롤린 뒤에 제외) 리신과 아르기닌 후 실리 트립신 소화를 수행합니다. </li><li> 변수 수정 및 고정 수정과 같은 시스테인 잔기의 carboxyamidomethylation (57.021464 다)와 같은 산화 된 methionines (15.99491 다)를 허용하는 데이터베이스 검색 매개 변수를 설정합니다. 마스코트 우리를 검색을위한고해상도 스캔을 보내고, 15 ppm으로 전구체 물질 허용 오차를 설정하고 다 0.6 단편 질량 허용 오차를 설정합니다. 마지막으로, 1 %에 단백질 FDR을 설정합니다. </li><li> P.의 마스코트 검색을 가져 오기 비계 (Proteomesoftware, 버전 3)에 루기 노사 CCMS 실험은, 1 % 단백질 FDR 가까이를 취득하고, 총 스펙트럼 수를 추출하는 매개 변수를 설정합니다. </li><li> 대표적인 결과는 본 논문에서는, 우리는 경쟁 제어 실험을 비교하는 쌍 T 테스트를 사용하고, 0.1 이하의 p 값, 2 (스펙트럼 수를 실험 / 경쟁 제어 스펙트럼 위의 스펙트럼 카운트 비율 안타를 고려 카운트). </li><li> 데이터는 추가 분석을 위해 스프레드 시트 소프트웨어에서 내보낼 수 있습니다. </li></ol> 10. 레이블이없는 정량화 <ol><li> Progenesis LC-MS 소프트웨어 (비선형 역학, 버전 4.0)에 원시 파일을 가져옵니다. </li><li> 기본 setti에서 LC-MS 배향 및 특징 검출을 수행NGS. </li><li> Progenesis LC-MS에서 MGF 형식으로 데이터를 내 보냅니다. </li><li> NCBI P. 대하여 MASCOT를 사용 MS / MS 스펙트럼을 검색 앞으로 함유 역 미끼 단백질 항목 aeruginosa의 데이터베이스. </li><li> Progenesis LC-MS로 데이터베이스 검색 결과를 가져 오기 및 MS1 기능에 펩티드의 식별을 매핑합니다. </li><li> (0.1 이하, Q 값, 상기 2 스펙트럼 계수 비 임계) 데이터 평가 (유의 수준의 계산식 폴드 변경 비율)의 내용 SafeQuant ProteinSQAnalysis 스크립트를 사용 하였다. </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+diguanosine+monophosphate+represses+bacterial+flagella+synthesis+by+interacting+with+the+Walker+A+motif+of+the+enhancer-binding+protein+FleQ.">Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 18478-18483 (2013).</li><li>Nesper, J., Reinders, A., Glatter, T., Schmidt, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+capture+compound+for+the+identification+and+analysis+of+cyclic+di-GMP+binding+proteins.">A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.</a> J Proteomics. 75, 4874-4878 (2012).</li><li>Roelofs, K. G., Wang, J., Sintim, H. O., Lee, V. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+thermophoresis+quantifies+biomolecular+interactions+under+previously+challenging+conditions.">Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.</a> Methods. 59, 301-315 (2013).</li><li>Merighi, M., Lee, V. 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Chem. 286, 2910-2917 (2011).</li></ol>

The authors have nothing to disclose.

특별한주의가 프로토콜의 여러 단계에서주의해야한다. 단백질 농도가 10 ㎎의 농도를 갖는 중요한 파라미터이다 / ml의 세포가 특정한 성장 조건 (예를 들면 작은 콜로니 생물막 또는 변이체) 하에서 성장 될 때 도달하기 힘들어. 따라서, 펠릿 부유 용균 완충액 저용량에서 수행되어야한다. 단백질 농도는 8 ㎎ / ㎖로 감소 될 수있다. Nesper 외. (1)에 의해 게시 된 방법과 비교하?...

C 디 GMP 그들의 성장 및 행동의 다양한 측면을 제어하기 위해 사용되는 대부분의 세균에 의해 두번째 키 메신저. 예를 들어, C-디 GMP는 세포주기 진행, 운동성 및 엑소 폴리 사카 라이드 및 표면 adhesins 2-4의 발현을 조절한다. 이러한 프로세스의 조정을 통해 C 디 GMP는 biofilm 형성, 병원성 박테리아 5의 범위의 만성 감염과 관련된 프로세스를 촉진합니다. C 디 GMP는 촉매 GGDEF 도메인 4 항구 diguanylate cyclases (DGCs)라는 효소에 의해 synthetized된다. 일부 DGCs 다운 C-GMP 디 바인딩시 클라 제 활성을 조절하는 사이트 억제을 갖는다. C 디 GMP의 분해는 촉매 EAL 또는 HD-GYP 중 도메인 6,7 숨겨 포스 포의 두 가지 클래스 (PDE는)에 의해 촉매된다. 직접 C 디 GMP 결합 공지 효과기 단백질의 대부분은 단백질의 세 개의 클래스 중 하나에 속한다 : 촉매를동맹 비활성 GGDEF 또는 EAL 도메인과 필츠 (Pilz) 도메인, C 디 GMP 8 바인딩에 따라 구조 변화를 받아야 작은 분자 스위치. DGCs, PDE는 필츠 단백질은 잘 특징과 도메인은 상대적으로 안전하게 실리에 예측할 수있다. 특히 관심은 이제 C 디 GMP 이펙터의 새로운 클래스의 식별에 초점을 맞추고 있습니다. 다른 바인딩 모티브 일부 C 디 GMP 이펙터는 부르크 홀데 리아 cenocepacia에서 CRP / FNR 단백질 가족 Bcam1349 또는 P.의 전사 조절 FleQ로 최근에 이러한 설명했다 녹농균 (9, 10). 또한, C-GMP 디 특정 리보 스위치 최근 식별과 C 디 GMP 의존적 11 유전자 발현을 제어하는 것으로 나타났다. 다른 이펙터의 C 디 GMP 바인딩 모티브는 제대로 보존 된 단백질의 생물 정보 학적 예측을 어렵게하고있다. 이 문제를 해결하기 위해, 우리는 C 디 GMP의 SPE의 사용에 기초 생화학 방법을 개발cific 캡처 화합물은 질량 분석 1,12,13과 함께. 우리는 최근 (CDG-CC, 그림 1) 새로운 가의 C 디 GMP 캡처 화합물을 설계 한 일. 이 화학 골격이 구성된다 결합 단백질 C 디 GMP를 캡처 미끼로 이용 1) C 디 GMP 잔기, 2) UV-광활성 반응성 기는 링크 크로스를 사용 결합 단백질 CDG-CC 및 3) 비오틴 스트렙 타비 딘 - 코팅 된 자성 비드를 이용하여 캡쳐 된 단백질을 분리. CDG-CC 직접 구체적 세포 용 해물과 같은 거대 분자의 복합체 C로부터 디 GMP 이펙터를 캡쳐 할 수있다. 캡처 계 화합물 및 화학 프로테오믹스 기반 방식은 이전 생물 광범위한 적용 것으로보고 된 예 Caulobacter crescentus, 살모넬라 엔테 혈청 형 티피 뮤 리엄 및 P. aeruginosa의 1,14. 이 방법론 논문에서,우리는 P. 추출물을 사용 CCMS 절차의 깊이 설명에 제공 예를 들어 녹농균. 이 연구는 생화학 적으로 작은 분자 신호 전달에 관여하는 새로운 구성 요소를 식별 할 수있는 강력하고 다양한 도구로 CCMS를 설정합니다.

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

상당한 진전은 제 메신저 C 디 GMP의 합성 (diguanylate cyclases) 열화 (포스 포)에 관여하는 효소의 확인 및 특성화를 향해 지난 10 년 동안 만들어졌다. 대조적으로, 약간의 정보가이 시그널링 분자가 세포 과정의 다양한 범위를 조절하는 분자 메커니즘을 통해 세포 성분과 관련하여 가능하다. 알려진 이펙터 단백질의 대부분은 필츠 (Pilz) 가족에 속한다 또는 촉매를 포기하고 이펙터 기능을 채택했다 diguanylate cyclases 또는 포스 포 디에스 퇴화된다. 따라서, 더 나은 실험 방법은 실리코 예측의 신뢰성이 실패하는 신규 이펙터를 식별하고 검증하는 데 필요한 박테리아 광범위 셀룰러 C 디 GMP 네트워크를 정의한다. 우리는 최근에 강력한 도구로 새로운 캡처 복합 질량 분석 (CCMS) 기반 기술을 개발했다생화학 적 식별 및 C 디 GMP 결합 단백질을 특성화. 이 기법은 이전에 하나의 유기체에 적용 가능한 다양한 것으로보고되었다. 여기서 우리는 이러한 신호 성분을 프로빙 이용하는 프로토콜의 자세한 설명을 제공한다. 예로서, 우리는 녹농균, C 디 GMP는 병독성 및 생물막 제어에 중요한 역할을하는 기회 적 병원균 사용. CCMS는 C 디 GMP 네트워크의 공지되거나 예측 된 성분의 74 % (51분의 38)를 식별. 본 연구는 상세히 CCMS 절차를 설명하고, 소분자 시그널링에 관련된 신규 한 구성 요소를 식별하기위한 강력하고 다양한 도구로 정한다.

P.에 새로운 C 디 GMP 이펙터를 확인하려면 aeruginosa에 우리는 체계적으로 P.의 가용성과 막 분수를 분석하는 CCMS를 사용 로그 상 문화 루기 노자 균주 PAO1 (OD = 0.5 (600)). 여기에서 우리는 요약하고이 낚시 원정대의 대표적인 결과에 대해 설명합니다. 네 개의 독립적 인 생물 복제를 사용 하였다. 각 실험을위한 두 가지 CDG-CC 농도 (5 μM 및 10 μM)을 사용 하였다. 특이성...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

캡처 복합 질량 분석 - 소설의 C 디 GMP 이펙터 단백질을 식별 할 수있는 강력한 도구

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

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11.6K 조회수.  Biozentrum of the University of Basel. The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

비디오: Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

든 샘플 준비 : 울트라 씬 레이어 방법

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

연구

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 ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

전기 분무 이온화 질량 분광법을 사용하여 효모 Lipidome의 양적 평가

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

분할 Ubiquitin 기반 막 효모 두 하이브리드 (신화) 시스템 : 단백질 단백질 상호 작용을 확인을위한 강력한 도구

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

구조 질량 분광법에 의해 큰 단백질 단지 분석

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

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

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

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 ...

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 ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

펩타이드 면역 친 화성 농축을 사용하여 단백질의 양을 정함은 질량 분광법과 함께

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Pak2 활성화의 아미드 수소 / 중수소 교환 및 든 - TOF 질량 분광법 분석

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

유전자 발현의 DNA 상호 작용, 디스 전사 레귤레이터, 그리고 소설 안티 종양 에이전트를 식별 : 단백질을 연구하는 정량 분석

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

캡처 복합 질량 분석 - 소설의 C 디 GMP 이펙터 단백질을 식별 할 수있는 강력한 도구

요약

더 많은 비디오 탐색

이 비디오의 챕터

든 샘플 준비 : 울트라 씬 레이어 방법

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

전기 분무 이온화 질량 분광법을 사용하여 효모 Lipidome의 양적 평가

분할 Ubiquitin 기반 막 효모 두 하이브리드 (신화) 시스템 : 단백질 단백질 상호 작용을 확인을위한 강력한 도구

구조 질량 분광법에 의해 큰 단백질 단지 분석

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

Methyltransferases의 프로 파일링 및 기타 S - adenosyl -_{L - 호모 시스테인 결합 단백질}

펩타이드 면역 친 화성 농축을 사용하여 단백질의 양을 정함은 질량 분광법과 함께

Pak2 활성화의 아미드 수소 / 중수소 교환 및 든 - TOF 질량 분광법 분석

유전자 발현의 DNA 상호 작용, 디스 전사 레귤레이터, 그리고 소설 안티 종양 에이전트를 식별 : 단백질을 연구하는 정량 분석

캡처 복합 질량 분석 - 소설의 C 디 GMP 이펙터 단백질을 식별 할 수있는 강력한 도구

요약

더 많은 비디오 탐색

이 비디오의 챕터

든 샘플 준비 : 울트라 씬 레이어 방법

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

전기 분무 이온화 질량 분광법을 사용하여 효모 Lipidome의 양적 평가

분할 Ubiquitin 기반 막 효모 두 하이브리드 (신화) 시스템 : 단백질 단백질 상호 작용을 확인을위한 강력한 도구

구조 질량 분광법에 의해 큰 단백질 단지 분석

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

Methyltransferases의 프로 파일링 및 기타 S - adenosyl - L - 호모 시스테인 결합 단백질

펩타이드 면역 친 화성 농축을 사용하여 단백질의 양을 정함은 질량 분광법과 함께

Pak2 활성화의 아미드 수소 / 중수소 교환 및 든 - TOF 질량 분광법 분석

유전자 발현의 DNA 상호 작용, 디스 전사 레귤레이터, 그리고 소설 안티 종양 에이전트를 식별 : 단백질을 연구하는 정량 분석

Methyltransferases의 프로 파일링 및 기타 S - adenosyl -_{L - 호모 시스테인 결합 단백질}