이 작품은 건강 교부금 GM086822과 국립 과학 재단의 주요 연구 계장 보조금 DBI-0960313 국립 연구소에 의해 재정 지원되었다. 저자는 Drs를 감사드립니다. 존 미야케 &amp; 도나 하디 (바이오 방사선)과 기술적 전문성을위한 제니퍼 Loertscher (시애틀 대학). 크로마 토그래피의 시약을 선택하고 추가 장비가 넉넉한 바이오 방사선에 의해 제공되었다.

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크로마 토그래피의 시약을 선택하고 추가 장비는 바이오 - 방사선에 의해 제공되었다.

액체 크로마 토그래피 기법 7 생화 학적, 면역 6을 수행하는데 필요한 고도로 정제된 단백질을 준비하고, 구조적 8 연구를 위해 귀중한를 입증했습니다. 혼성 집적회로 정화 방법은 가장 많이 선호하는 매체의 경험적 결정을 요구하고, 리간드 구조, 리간드 밀도, 그리고 매트릭스 비드 속성은 모든 색층 결과 2, 3에 영향을 보여줘왔다. 정찰 자동 열은 이후의 최적화 및 단백질 정제 4 혼성 집적회 매체 선택을위한 효율적인 접근 방법입니다. 제시 자동화된 컬럼 탐색 방법을 쉽게 다양한 혼성 집적회로 단백질 정제 전략에 적용할 수 있습니다. 소금 농도, 소금의 선택, 소금 그라데이션, 그리고 산도의 변경은 더 정화 조건을 향상시킬 수 있으며, 이러한 필수 매개 변수를 변화의 효과는 이전에 13,14을 검토하고있다. 부분적으로 정화 대상 prote를 포함하는 혼성 집적회로 eluate안에는 더 이상 이러한 이온 교환 크로마 토그래피 (IEX) 또는 겔 여과 / 크기 배제 크로마 토그래피 9,10과 같은 보완 색층 기법을 사용하여 투석을 할 수 있습니다. 워낙 독특한 조명 흡광도 및 방출 특성 때문에, GFP의 용출 프로파일은 인라인 및 사후 운영하는 방식을 사용하여 확인할 수 있습니다. 이를 위해,이 프로토콜은 더 이상 관련이없는 대상 단백질이 GFP와 &quot;태그가&quot;되는, 재조합 GFP-융합 단백질의 정제에 맞게 조정 할 수 있습니다. GFP-태그가 대상 단백질은 자외선 11 위에서 설명한 혼성 집적회로 정화 전략 등 다양한 색층 접근법을 사용하여 정제로 감지가 가능하다. GFP 정화는 또한 현대 단백질 과학 기술 12 교육을위한 생화학 실험실에서 교육학 주식이되었다. 기본적인 혼성 집적회로 단백질 정제는 크게 덜 강력한 크로마 토그래피 싸이를 사용하여 수행할 수 있지만줄기, 여기서 제시된 계측이 유리한 결과를 얻기에 투입하기 위해 뚜렷한 장점을 가지고 있습니다. 고도로 자동화된 시스템을 활용의 주목할만한 장점이 실행 대 실행 조건 재현성, 시간 절감 효과를 개선하고, 시스템 10에 소개되는 공기의 기회를 감소 등이 있습니다. 시스템 극대화에 의해 촉진되고 시스템 제어 버퍼 블렌딩은, 버퍼 준비 및 실험 재현성의 향상 일관성을 허용합니다. 모든 정찰을위한 동적 샘플 루프에 충분한 샘플 하중을 설계하고 실행하는 것은 더욱 각 컬럼에 추가되는 샘플의 균일을 보장하고 중단하거나 다시로드 매뉴얼없이 순차적인 실행을 허용합니다. 칼럼쪽으로 샘플 루프에서 제어 샘플 주입 수동 샘플 로딩과 함께 발생할 수있는 다양성을 낮춥니다. 열 선택 밸브의 쌍은 시스템을 replumb 필요없이 연속 샘플 실행, 다른 혼성 집적회로 컬럼을 사용하여 각각에 대해 허용합니다. mul 공연<html> 독특한 분광 프로필로 단백질을 시금 때 TI-파장 분석에 특히 유용합니다. GFP 외에도 cytochromes, flavoproteins 및 기타 헴 함유 단백질이 기술로부터 혜택을 누릴 수 있습니다. 전도율과 산도 모니터링 디바이스는 실시간 실험 조건의 확인을 위해 수 있습니다. 분할 분획 eluate 컬렉션은 향상된 분획 처리 가능하며 최소한의 샘플 볼륨 (예 : 엘리사, SDS-PAGE, 서부 얼룩 및 Experion microfluidic 전기 영동) 요구 이후의 운영 분석 방법에 대해 쉽게 전송이 가능합니다. 두 번째 분획 컬렉터 오프라인을 운영하는 것은 수동 동기화가 필요하지만, 그것은 분획 수집기 선택에 최대의 유연성을 허용합니다. 혼성 집적회로 정찰 칼럼 및 단백질 정제를 위해 이러한 강력한 크로마 토그래피 시스템을 활용하여 가장 중요한 단점은 초기 시간과 악기 수집 및 운영자 교육과 관련된 예산 지출을 포함합니다. T &quot;&gt; 여기를 제시 프로토콜 바이오 방사선 DuoFlow 크로마 토그래피 시스템을 활용,. 그러나, 이러한 GE 헬스케어에서 악타의 복고 등 다른 제조 업체,에서 똑같이 강력 계장도 활용 수 있으며, 이에 상응하는 결과를 생산하는 능력이있다하더라도 비교 크로마 토그래피 시스템 정화 절차 또는 악기 수집을 시작하기 전에 고려해야하는 고유의 특성 (예를 들면 메소드 프로그래밍, 컴포넌트 명명법, 사업자 환경, 그리고 확장성 제한)가.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">생물 학적 DuoFlow 탐험자 20 시스템</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">7,602,257</a> </td><td> 시스템의 극대화, 믹서, F10 워크 스테이션, AVR7-3 밸브, QuadTec의 UV / 흐름 전지, BioFrac 분획 수집기, 생물 학적 소프트웨어 VIS 검출기 및 스타터 키트 포함 </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">AVR9-8 스트림의 선택 밸브</a><html></td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7,600,408</a> </td><td> 생물 학적 DuoFlow 시스템에서 사용하기에 고압 밸브, 9 포트, 8 위치, 3,500 PSI (233 바) 한도, </td></tr><tr><td> 분배기 밸브 SV3T-2 </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> 7,600,410 </td><td> 생물 학적 DuoFlow 시스템을 함께 사용하기위한 솔레노이드 밸브, 3 포트, 2 순위, 30 PSI (2 바) 한도, </td></tr><tr><td> 스트림 스플리터 밸브 </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td></td><td></td></tr><tr><td>tical = LSR &amp; parentCategoryGUID = 22c495f1-36ff-44a3-9140-ee7e54d0b1ba &quot;대상 =&quot;_blank &quot;&gt; DynaLoop 25 키트 </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0451&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7,500,451</a> </td><td> 샘플 루프 키트 슬라이딩 - 피스톤은 피스톤 루프를 슬라이딩 25 ML의 DynaLoop, DynaLoop 부품 키트 (# 750-0450)가 포함되어 있습니다 </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">BioFrac 분수 수집기</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">7,410,002</a> </td><td> 두 분획 열<html> lectors 사용된 </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">BioFrac microplate 드롭 헤드 어댑터</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">7,410,088</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=741-0017" target="_blank">BioFrac microtiter 플레이트 어댑터</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td>&amp; 국가 = 미국과 랭 = ko를 &amp; 제품 일련 = 741-0017 &quot;대상 =&quot;_blank &quot;&gt; 7410017 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">자외선 광학 모듈</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">7,500,202</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-1331&vertical=LSR&parentCategoryGUID=c4095dc1-9260-4431-b066-823b050048c8" target="_blank">할로겐 램프</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> &lt;<html> 대상 = &quot;_blank&quot;&gt; 7601331 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">이코노 그라데이션 펌프</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">7,319,001</a> </td><td> 저압 단백질 정제를위한 기울기 펌프, 튜브 및 부속품 키트를 포함 </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experion 시스템</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td>bio-rad.com/prd/en/US/adirect/biorad?에 cmd = BRCatgProductDetail &amp; 제품 일련 번호 = 224301 &quot;대상 =&quot;_blank &quot;&gt; 7007001 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">Experion Pro260 분석 키트</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">바이오 방사선</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">7,007,102</a> </td><td></td></tr><tr><td> HiTrap 혼성 집적회로 열 선택 키트 </td><td> GE 헬스케어 </td><td> 28-4110-07 </td><td> 정찰을위한 혼성 집적회로 미디어 7 × 1 ML 미리 포장 열이 포함 </td></tr><tr><td> GE 헬스케어 - 투 - 바이오 - 방사선 칼럼 피팅 </td><td> GE 헬스케어 </td><td> 18111251 및 18111257 </td><td></td></tr></tbody></table>

automated hydrophobic interaction chromatography column selection for use in protein purification

정화 단백질 (예 : 젤 여과, 친화력, 그리고 이온 교환) 다른 색층 방법와는 달리, 소수성 상호 작용 크로마 토그래피 (혼성 집적회로)는 일반적으로 가장 적합한 색층 매체를 선택하기 위해 실험적 결정 (심사 또는 &quot;정찰&quot;로 칭함)가 필요합니다 주어진 단백질 1 정화입니다. 여기에 제시된 방법은 단백질 정제에 사용되는 최적의 혼성 집적회로 미디어를 찾는다는 뜻에 대한 자동 접근 방식을 설명합니다. 혼성 집적회로가 소수성의 차이를 기반으로 원유 lysate에서 단백질 및 기타 biomolecules를 분리시킵니다. 친화성 크로마 토그래피 (AC)와 이온 교환 크로마 토그래피 (IEX)와 마찬가지로, 혼성 집적회는 색층 프로세스를 통해 진행됨에 따라 관심 단백질을 집중시킬 것입니다. 최고의 혼성 집적회로에 의해 정화에 적합 단백질은 소수성 표면 영역 및 2 M의 탄약을 초과하는 농도를 방해하기 위해 노출을 견딜 수있는 사람을 포함nium 황산 ((NH 4) 2 SO 4). 혼성 집적회로 종종 친화 태그가 부족한 단백질을위한 정제 방법으로 선택하고, 교류를위한 따라서 적합하고, IEX는 충분한 정화를 제공하는 데 실패하면된다. 단백질 표면의 소수성 moieties 일시적으로 불활성, 움직이기 힘드는 매트릭스를 결합 무극 성 리간드에 바인딩. 단백질과 리간드 사이의 상호 작용이 단백질 - 리간드 상호 작용을 강화하고 움직이기 힘드는 단백질 (즉, 열은 내부 구속) 2를 만들기 높은 이온 농도로, 크로마 토그래피 칼럼을 통해 흐르는 버퍼의 소금 농도에 크게 의존합니다. 소금 농도 감소로 단백질 리간드 상호 작용 없어져요, 단백질 다시 컬럼에서 모바일 및 elutes된다. 여러 혼성 집적회로 미디어가 미리 포장 컬럼에서 상업적으로 사용할 수있는 여러 소수성 리간드 (예 : octyl S-부틸, 부틸, 그리고 페닐) 각각 포함하는 하나 사양의 아가로 오스 비즈로 밀도를 변화에서 크로스는-연결된ific 직경 3. 정찰 자동 열은 혼성 집적회로 미디어가 앞으로 더 철저한 최적화 실험을 위해 고용 및 단백질 정제 4를 운영해야하는 결정하기위한 효율적인 접근을 허용합니다. 여기를 정화하는 특정 단백질은 재조합 녹색 형광 단백질 (GFP)입니다 있으나, 접근은 하나 이상의 소수성 표면 영역과 다른 단백질 정화에 맞게 조정될 수 있습니다. GFP가 자외선 5로 노출시의 안정성, 397 nm의에서 독특한 빛의 흡광 피크, 그리고 형광에 의한 유용한 모델 단백질로서의 역할을합니다. 야생 형 GFP를 포함하는 세균성 lysate는 높은 소금 버퍼에 준비된 바이오 방사선 DuoFlow 매체 압력 액체 크로마 토그래피 시스템에로드하고, 다른 혼성 집적회로 미디어를 포함하는 HiTrap 혼성 집적회로 컬럼에 adsorbed되었다. 단백질은 칼럼에서 eluted과 인라인 및 사후 운영하는 검출 방법에 의해 분석되었다. 버퍼 블렌딩, 동적 시료 루프 주입, 순차 열umn 선택, 다중 파장 분석 및 분할 분획 eluate 컬렉션은 실험적 접근 방식의 시스템과 재현성의 기능을 향상시켰습니다.

Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

단백질 정제의 과정에서 사용될 적합한 소수성 상호 작용 크로마 토그래피 (혼성 집적회로) 미디어를 식별하는 자동화된 방법을 설명하고있다. 방법은 자동화된 버퍼 블렌딩, 동적 시료 루프 주입, 순차 열 선택, 다중 파장 분석 및 분할 분획 eluate 컬렉션을 포함하여 중간 압력 액체 크로마 토그래피 시스템을 활용합니다.

단백질 정제에서 사용하기 위해 자동화된 소수성 상호 작용 크로마 토그래피 칼럼 선정

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

automated-hydrophobic-interaction-chromatography-column-selection-for

Research

JoVE Journal

Biology

43.1K 조회수.  Seattle University. 단백질 정제의 과정에서 사용될 적합한 소수성 상호 작용 크로마 토그래피 (혼성 집적회로) 미디어를 식별하는 자동화된 방법을 설명하고있다. 방법은 자동화된 버퍼 블렌딩, 동적 시료 루프 주입, 순차 열 선택, 다중 파장 분석 및 분할 분획 eluate 컬렉션을 포함하여 중간 압력 액체 크로마 토그래피 시스템을 활용합니다.

비디오: Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

연구

생물학

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

의 정화 M. magneticum 스트레인 AMB - 1 Magnetosome 관련 단백질 MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

헬리 우스 진 건을 사용하여 양파 세포에서 단백질 - 단백질 상호 작용에 대한 Bimolecular 형광 Complementation (BiFC) 어세이

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

자기 활성 셀 정렬 (LIMACS)를 사용하여 지질 소포 - 중재 동질 크로마 토그래피 : 단백질 지질 상호 작용을 분석하는 소설 방법

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104의 정화, 단백질 Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

재조합 단백질의 미끼를 사용 파지 디스플레이와 궁합 선택을위한 프로토콜

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

단백질 - 단백질 상호 작용을 확인<em&gt; 초파리</em탠덤 선호도 정화로&gt; 성인 머리 (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

펩타이드 배열을 사용하여 단백질 - 단백질 상호 작용 사이트를 확인

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

살아있는 세포의 단백질 조각 보완 분석 (PCA)에 의해 게놈 전체 단백질 - 단백질 상호 작용 상영

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase 보완성 이미징 분석 결과 Nicotiana benthamiana 잎 뚜렷이 결정 단백질 단백질 상호 작용 역동성에 대 한

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...