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

Nanyang Technological University

Research

JoVE Journal

Biology

43.4K 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

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetotate 하이드로 아세테이트 도메인 함유 단백질의 발현, 정제, 결정화 및 효소 어세포

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

연구

생화학

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

다중 컬럼 플레이트 어댑터를 사용하는 경제적이고 다재다능한 고처리량 단백질 정화 시스템

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
<div class="chapter_text" id="script_0">
&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
</div>
<div class="chapter_text" id="script_1">
Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
</div>
<div class="chapter_text" id="script_2">
To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
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<div class="chapter_text" id="script_3">
Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

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In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
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Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
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You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Size Exclusion and Affinity Chromatography

크로마토그래피 기반 생체 분자 정화 방법

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

교육

JoVE Science Education

화학

Ion-Exchange Chromatography

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ion-exchange chromatography is a type of chromatography that separates analytes based on charge. A column is used that is filled with a charged stationary phase on a solid support, called an ion-exchange resin. Strong cation-exchange chromatography preferentially separates out cations by using a negatively-charged resin while strong anion-exchange chromatography preferentially selects out anions by using a positively-charged resin. This type of chromatography is popular for sample preparation, for example in the cleanup of proteins or nucleic acid samples.
Ion-exchange chromatography is a two-step process. In the first step, the sample is loaded onto the column in a loading buffer. The binding of the charged sample to the column resin is based on ionic interactions of the resin to attract the sample of the opposite charge. Thus, charged samples of opposite polarity to the resin are strongly bound. Other molecules that are not charged or are of the opposite charge are not bound and are washed through the column. The second step is to elute the analyte that is bound to the resin. This is accomplished with a salt gradient, where the amount of salt in the buffer is slowly increased. Fractions are collected at the end of the column as the elution occurs and the purified sample of interest can be recovered in one of these fractions. Another technique, such as spectroscopy, may be needed to identify which fraction contains the sample. Ion-exchange chromatography is especially useful in protein studies, to isolate proteins of interest that have a specific charge or size, as size can determine the number of interactions with the resin.
Ion-exchange chromatography is a more general separation technique than affinity chromatography, which is also often used in preparing protein samples, where an antibody is attached to a column to bind one specific analyte. A new affinity column must be purchased for each analyte, while the same type of ion-exchange column, often with different eluting conditions, can be used to clean up many proteins of the same charge. Ion-exchange chromatography can also be used in conjunction with other types of chromatography that separate based on other properties. For example, size-exclusion chromatography separates based on size and could be used before ion-exchange chromatography to choose compounds of only a given size.

Ion-Exchange Chromatography: Principle and Protein Separation

이온 교환 크로마토그래피

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ion-exchange chromatography is a type of chromatography that separates analytes based on ...

분석 화학

Hydrophilic Interaction Liquid Chromatography: A Technique to Separate Hydrophilic Polar Analytes Using Hydrophilic Beads

Source: Liu, D., et al. <a href="https://www.jove.com/v/60104">Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography</a>. J. Vis. Exp. (2020).
This video demonstrates the principle of separation of hydrophilic polar compounds using hydrophilic interaction liquid chromatography. This technique helps in the purification of various polar analytes, including N-glycans.

친수성 상호 작용 액체 크로마토그래피: 친수성 비드를 사용하여 친수성 극성 분석물을 분리하는 기술

Source: Liu, D., et al. Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography. J. Vis. Exp. (2020).
This video demonstrates the ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

단클론 항체 정제를 위한 친화성 크로마토그래피 기법

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

Immunology

Antibody-Based Technologies

Production and Purification

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

Source: Karch, C. P. et al., <a href="https://app.jove.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

Affinity Chromatography를 사용한 자체 조립 단백질 나노 입자의 정제

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

Immunotherapy

Immunogenics and Vaccine Development

Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

Source: Oller-Salvia, B. <a href="https://www.jove.com/v/58066">Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction</a>. J. Vis. Exp. (2018).
This video demonstrates the hydrophobic interaction liquid chromatography-based characterization of antibody-drug conjugates. Using a butyl ligand-based chromatography column, the antibody-drug conjugates are characterized via hydrophobic interactions with the ligand.

Butyl-Dedependent Hydrophobic Interaction Liquid Chromatography: 소수성 상호작용을 기반으로 항체-약물 접합체를 특성화하는 기법

Source: Oller-Salvia, B. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal ...

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography 
When the protein&#39;s chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.
Ion Exchange Chromatography
Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged &#8212; positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.
Affinity Chromatography
Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the&#160; column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-&#946;-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.
High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

컬럼 크로마토그래피의 종류

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating ...

JoVE Core

Cell Biology

Unit 1

Analyzing Cells and Proteins

주요 용어 및 개념

Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Start the FPLC with general maintenance. Connect column buffers AIEX-A and AIEX-B to buffer valves, and the anion-exchange resin-containing column to the column ports of the FPLC. Adjust the general chromatographic parameters accordingly.
Start the AIEX program in the FPLC software and choose the equilibration volume for buffer A. Subsequently, load the sample onto the column, and elute the proteins with a linear gradient from 0% to 100% high-salt buffer. Collect 2-milliliter fractions during elution.
Analyze the eluted fractions after finishing the run.