Name: in vitro analysis of pdz-dependent cftr macromolecular signaling complexes
Uploaded: 2012-08-13T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

우리의 작품은 미국 심장 협회 (대동 동남 제휴) 초급 - 보조금의 0765185B, 엘사 미 Pardee 재단 연구 기금, 그리고 웨인 주립 대학교 교내 시동 펀드, 심장 혈관 연구소 이시스 전략 어워드에서 교부금에 의해 지원되었습니다. 체외 CFTR의 macromolecular 복합 어셈블리에서이 방법은 원래 박사는 AP 통신 Naren (테네시 보건 과학 센터의 대학)에 의해 개척되었다.

1. 박테리아의 재조합 태그 퓨전 단백질의 표현과 정제 <ol><li> CFTR, LPA 2, MRP2, MRP4, β 2 AR, 그리고 NHERFs (전체 길이 또는 PDZ1 또는 PDZ2 도메인을위한 C-꼬리 (C-말단에 PDZ 모티프를 포함하는 마지막 50-100 아미노산)의 정의 영역을 확대 ) PCR 방식에 의한. </li><li> GST-융합 단백질에 대한 pGEX4T-1 벡터 (예 : GST-NHERFs, GST-MRP4 중부 표준시와 같은), MBP-융합 단백질에 대한 pMAL-C2 벡터 (예 : MBP-β <s...

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이 프로토콜에서 우리는 이전에 보고된 16-19,29,30의 체외은 조립 및 정화 단백질 (혹은 단백질 조각) 및 / 또는 세포 lysates를 사용 macromolecular 신호 복잡한을 포함 CFTR의 검출을위한 방법을 보여주었다. 준비 과정에서 최상의 결과들에게 다음과 같은 중요한 사항을 달성하기 위해서는 특별한주의가 필요합니다 : <ul><li> 그것은 용출 버퍼의 산도가 단계 1)에서 설명한 GST-융합 단백질...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> pGEX4T-1 벡터 </td><td> GE 헬스케어 </td><td> 28-9545-49 </td><td> 이전 Amersham Biosciences </td></tr><tr><td> pMAL-C2 벡터 </td><td> 뉴잉글랜드 BioLabs </td><td></td><td></td></tr><tr><td> pET30 벡터 </td><td> EMD 화학 </td><td> 69077-3 </td><td> 이전 Novagen </td></tr><tr><td> 글루타티온 아가로 오스 비즈 </td><td> BD Biosciences </td><td> 554,780 </td><td></td></tr><tr><td> 아밀로오스 수지 </td><td> 뉴잉글랜드 BioLabs </td><td> E8021S </td><td></td></tr><tr><td> 탈론 비즈 </td><td> Clontech </td><td> 635,501 </td><td></td></tr><tr><td> 감소 글루타티온 </td><td> BD Biosciences </td><td> 554,782 </td><td></tD&gt; </tr><tr><td> 이미다졸 </td><td> 어부 </td><td> BP305-50 </td><td></td></tr><tr><td> 말토오스 </td><td> 어부 </td><td> BP684-500 </td><td></td></tr><tr><td> S-단백질 아가로 오스 </td><td> EMD 화학 </td><td> 69704-3 </td><td> 이전 Novagen </td></tr><tr><td> 안티 - 플래그 HRP </td><td> 시그마 </td><td> A8592 </td><td></td></tr><tr><td> 안티 CFTR IgG </td><td> 맞춤 양복 </td><td> R1104 </td><td> AA 722-734에서 CFTR 에피토프을 인식 mAb </td></tr><tr><td> 안티 MRP2 IgG </td><td> Chemicon 국제 </td><td> MAB4148 </td><td> Millipore의 지금 일부분 </td></tr></tbody></table> 표 2. 특정 시약 및 장비.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

낭성 섬유증의 transmembrane의 전도성 조정기 (CFTR), 주로 상피 세포의 꼭대기의 세포막에있는 염화물 채널, 1-3 transepithelial 유체 항상성에 중요한 역할을 담당하고 있습니다. 낭성 섬유증 (CF) 4 분비 설사 5 : CFTR은 두 가지 주요 질병에 연루되었습니다. CF에서 CFTR 망할 CIA 채널의 합성 또는 기능적 활동이 줄어 듭니다. 이 질환은 미국에서 2500 Caucasians 년 6 약 1에 영향을 미칩니다. 과도한 CFTR 활동도 캠프의 용기 7의 cGMP 생산을 자극 독소 유발 분비 설사 (예 : 콜레라 독소와 열 안정 E. 대장균의 enterotoxin에 의한)의 경우에 연루되었습니다. 증거를 수집하는 것은 CFTR 및 전송기, 이온 채널, 수용체, kinases, phosphatases, 신호 등 다른 단백질의 증가, 간의 물리적 및 기능적 상호 작용의 존재를 제안ING 분자, 그리고 cytoskeletal 요소 및 CFTR와 바인딩 단백질 간의 이러한 상호 작용은 비판적으로 시험 관내 및 생체내 또한 8-19에서 CFTR-매개 transepithelial 이온 전달을 조절에 관여하는 표시되었습니다. 이 프로토콜에서는, 우리는 방법에 초점을 그 단백질 바인딩 모티브를 가지고 CFTR 카르 복실 말단 꼬리 사이의 상호 작용 연구 지원과 [모티프의 PSD95/Dlg1/ZO-1 (PDZ)로 언급] 특정 바인딩 모듈을 포함하는 비계 단백질의 그룹 PDZ 도메인로 지칭. 지금까지 여러 가지 PDZ 비계 단백질 같은 NHERF1 등 다양한 동질성, NHERF2, PDZK1, PDZK2, CAL (CFTR-연관된 리간드), Shank2으로 CFTR의 카르 복실 말단 꼬리에 바인딩하고, 20-27를 파악하는 것으로보고되었습니다. PDZ 비계 단백질에 의해 인식되는 CFTR 사이 PDZ의 모티브는 C 말단 (즉, 인간의 CFTR에있는 1477-DTRL-1480) 20에서 마지막 네 아미노산이다. 흥미롭게도,CFTR은 동질성에게 22 변화와 함께이라도 NHERFs 및 PDZK1 모두 하나 이상의 PDZ 도메인을 바인딩할 수 있습니다. CFTR 구속력와 관련해서이 multivalency는 PDZ 비계 단백질은 세포 16-18에서 선택 / 특정하고 효율적인 신호를위한 복합 신호 CFTR의 macromolecular 형성을 촉진있다는 것을 암시, 기능성 중요한 것으로 표시되었습니다. 여러 생화학 assays는 공부를하기 위해 개발되었습니다 CFTR - 참여 등 공동 immunoprecipitation, 풀다운 분석, 페어 많다는 구속력을 분석, colorimetric 한쌍이 많다는 구속력을 분석하고, macromolecular 복잡한 어셈블리 분석 16-19,28,29 같은 단백질 상호 작용, . 여기 단백질 단백질이나 CFTR 16-19,28,29 관련된 도메인 도메인 상호 작용을 연구하기위한 실험실에 의해 광범위하게 사용되는 체외에서 복잡한 PDZ 모티브 종속적인 CFTR 함유 macromolecular 조립의 세부 절차에 초점.

Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

낭성 섬유증의 transmembrane의 전도성 조정기 (CFTR), 상피 염화물 채널, 각종 단백질과 상호 작용하고 중요한 세포 과정을 조절하는 것으로보고되었습니다; 그들 가운데 CFTR PDZ 모티프-매개 상호 작용이 잘 문서화되었습니다. 이 프로토콜은 우리가 복잡한 신호 PDZ-의존 CFTR의 macromolecular를 조립하기 위해 개발된 방법을 설명합니다<em&gt; 체외에서</em&gt;.

PDZ-의존 CFTR의 Macromolecular 신호 단지의 체외 분석

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

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In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

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