저자 방사성 레이블 바이러스의 준비를 위해 제약 생명 공학 및 분자 생물학의학과, 특히 모니크 드 Pelsmacker의 직원을 감사드립니다. 우리는 엘렌 Merckx하고 흥미로운 발언 및 토론을위한 Hadewych Halewyck과 실험실에서 그의 도움을 Gerrit 드 Bleeser에 감사를드립니다. 이 작품은 재정적 Vrije Universiteit Brussel (OZR1807), Fonds voor Wetenschappelijk Onderzoe​​k Vlaanderen (G.0168.10N)와 세계 보건기구 (TSA 200,410,791)의 OZR의 교부금에 의해 지원되었다. 리자 Schotte는 Fonds voor Wetenschappelijk Onderzoe​​k Vlaanderen (FWO)의 predoctoral 동료입니다.

원칙적으로 (A)와 방법에 대한 개요는 (B) 그림 1에 표시됩니다. 1. 버퍼의 작성 <ol><li> 용해 나트륨 dihydrogen 인산 (50 ㎜)과 물에 나트륨 염화물 (300 ㎜)에 의해 워시 / 바인딩 버퍼를 준비하고 8.0로 산도를 조정합니다. 또한 메티오닌 (2 % (M / V) 최종 농도) 및 알부민 십대 초반 20 (0.01 % (M / V) 최종 농도), (0.1 % (M / V) 최종 농도)을 추가하고 필요한 볼륨으로 조정합니다. </li></ol> 2. 마그네틱 비즈의 작성 사용 설명서에 따라 자기 구슬을 준비합니다. 간단히 : <ol><li> 와동을 사용하여 자기 구슬을 Resuspend. </li><li> 양도, 각 샘플에 대해, 튜브로 resuspended 자석 구슬 (40 밀리그램 / ML) 10 μl. </li><li> 뜨는가 분명하다 때까지 자석을 사용하여 자기 구슬을 모아서 (+ / - 30 초)과 뜨는을 조심스럽게 제거합니다. </li><li> 씻으십시오자석 구슬로 두 번 : 워시 / 바인딩 버퍼 150 μl에 구슬을 resuspend. 자석 구슬 뜨는 분명히 때까지 자석을 사용하여 튜브의 한쪽으로 마이 그 레이션하고 신중하게 뜨는을 제거합니다. </li><li> 구슬을 (10 밀리그램 / ML) resuspend하려면 각 샘플에 대해 워시 / 바인딩 버퍼 40 μl를 사용하십시오. </li></ol> 3. 동질 캡처 분석 참고 : (우주) 배경 방사능 (0 % 방사능)를 측정하려면 어떤 radiolabeled 바이러스가 제어 예제 1에 추가되지 않습니다. 대신, 같은 볼륨 바인딩 / 워시 버퍼가 추가됩니다. 참고 : 100 % 방사능 모든 구성 요소가 nanobody 제외하고는 추가됩니다하도록 컨트롤 샘플 2로 정의됩니다. 대신, 같은 볼륨 바인딩 / 워시 버퍼가 추가됩니다. <ol><li> 96 - 웰 microtiter 접시에 80 μl로 워시 / 바인딩 버퍼에 희석 (β-방사선) poliovirus 세이빈 변형 유형 1 9, 라벨 35 S 2000 CPM 와요. </li><li> 추가 10 &amp; MU; 우물에 nanobody 희석의 리터. </li><li> 흔드는를 사용하여 약 10 초간 섞는다. </li><li> 샘플은 실온에서 1 시간 품어하도록 허용합니다. </li><li> 한물간 자석 구슬 서스펜션의 40 μl를 추가하고 지속적으로 흔들어 아래 실온에서 10 분 동안 품어. </li><li> 구슬과 자석을 사용하여 표면에 뜨는을 분리하고 튜브로 정리 뜨는을 전송. </li></ol> 4. 방사능의 측정 <ol><li> 계산 플라스크에 단계 3.6에서 뜨는 50 μl를 전송합니다. </li><li> 섬광 액과 혼합 3 ML을 추가합니다. β-신틸레이션 계수기의 방사능을 측정합니다. </li></ol> 5. 비즈를 재활용 <ol><li> 이분 500 XG이나 맑은 뜨는 얻을 때까지 튜브와 원심 분리기에 사용되는 자성 비즈를 수집합니다. </li><li> 뜨는 제거 및 6 ML 0.5 M NaOH에 구슬을 resuspend. </li><li> multipl로 정학을 전송전자 튜브 5 분간 초음파 욕조에서 품어. </li><li> 자석 구슬을 수집하고 뜨는을 제거 자석을 사용합니다. </li><li> 각 관 1 ML 2% SDS-솔루션을 추가하고 와동을 사용 resuspend. 오분 동안 끓여야. </li><li> 비즈를 수집하고 뜨는을 제거하십시오. 한 ML 0.2 M EDTA (에틸렌 다이아 민 테트라 초산) (산도 = 7)에서 구슬을 Resuspend. </li><li> 5 분간 초음파 욕조에서 튜브를 부화하고 한번 더 뜨는을 제거합니다. </li><li> 각 튜브에 물 1 ML을 추가하고 염주를 resuspend. 비즈를 수집하고 뜨는을 제거하십시오. 두번 세차 단계를 반복합니다. </li><li> 뜨는을 제거하고 1 ML 10 밀리미터 CoCl 2 용액에 구슬을 resuspend. 10 분 동안 흔드는 쓰세요. </li><li> 비즈를 수집하고 워시 / 바인딩 버퍼의 1 ML에 resuspend하기 위해 자석을 사용하여 표면에 뜨는을 제거합니다. </li><li> 표면에 뜨는를 제거하고 비즈 20 %의 한 ML을 사용하여 두 번 (V / V) 에탄올을 씻어 </li><li> 20 % 원래 부피 (V / V) 동부 표준시에 구슬을 Resuspend40 MG / ML의 농도로 거듭난 구슬을 얻는 hanol. </li></ol> 6. 결과의 해석 <ol><li> 더 radiolabeled 바이러스가 추가되지 않았습니다있는 컨트롤 샘플 1, 배경 방사능을 위해 수정하고 0 % 방사능 값을 설정하는 데 사용됩니다. nanobody, 어디 따라서 모든 radiolabeled 바이러스가 뜨는에 남아를 포함하지 않는 컨트롤 샘플 2, 100 % 방사능 값으로 설정됩니다. </li><li> nanobody 의해 radiolabeled 바이러스 - 뜨는의 방사능의 비율 (= %) 전체 강수량 (% = 100)의 통계입니다. </li></ol> 7. 대표 결과 이 친화 캡처 분석을위한 대표적인 결과는 그림 2, 3 및 4에 표시됩니다. 그림 2에서 PVSS38C, poliovirus를위한 nanobody의 특정의 친화력이 나타납니다. PVSS38C의 친화력은 poliov 세 가지 항원에 대한 검사를했습니다irus : 네이티브-항원 (N-항원), 온수-항원 (H-항원)와 14S subunits. N-항원은 전염성이있는 그대로 바이러스입니다. 바이러스가 가열 (56 빠르면 20 분정 ° C) 또는 바이러스 RNA의 (부분) 손실 및 capsid 단백질 VP4 그 결과 고체 표면 (미발표 결과), capsid 변화의 형태로 부착하고, 비어있는 경우 capsids 그런 다음, H 항원이라고하는 형성된다. 14S subunits는 어셈블리 중간체입니다. 이러한 항원은 예방 접종 10시 항체의 다른 세트를 인정하므로 다른 epitopes와 antigenic 사이트를 보유하고 있습니다. 그림에서 뜨는에서 발견된 방사능의 비율은 nanobody의 농도의 함수로 표현된다. 그것은 radiolabeled 14S subunits를 포함하는 시료의 표면에 뜨는에서 방사능이 nanobody PVSS38C의 증가 농도와 함께 저하되는 모습을 볼 수 있습니다. 이것은 poliovirus 14S subunits 죄수에게의 양을 증가로 번역될 수nanobody PVSS38C와 간접적으로 자석 구슬로 nected. 캡처 titer는 (radiolabeled 바이러스의 50 %를 캡처 nanobody 필요한 중 = 농도) 그래픽 0.099 NM로 계산됩니다. N-antigenic과 poliovirus의 H-antigenic 양식에 대한 PVSS38C의 어떠한 친화력을 준수하지 않습니다. 이러한 데이터에서 그것은 PVSS38C가 poliovirus의 두 개의 다른 antigenic 양식에 대한 독점적 14S subunit에 존재하며 없습니다 에피토프와 상호 작용하는 것을 해석됩니다. 표면에 뜨는에서 방사능의 손실만이 그 항원에 대한 nanobody의 구체적인 친화력에 의한 것이 입증하기 위해 동일한 분석이 Nb1으로 수행되었다 Sulfolobus sulfataricus의 lrpB transcriptional 레귤레이터로부터 생성되고 알려진 nanobody은 상호 작용과이 없습니다 모든 poliovirus의 항원. 이 분석의 결과는 그림 3에 표시됩니다. 모든 radiolabeled 바이러스는 전혀 반응이 없다는 것을 보여주는 supernatants에 남아 있습니다poliovirus의 세 antigenic 양식에 대한 Nb1. 결과의 재현성이 다른 일에 하나의 주어진 nanobody (즉, PVSP29F)에 여덟 배나 총 실험을 반복하여하고 다른 사람에 의해 테스트되었다. 결과는 그림 4에 표시됩니다. 뜨는의 비율 방사능의 평균 가치는 각 nanobody 농도에 대해 계산하고 표준 편차와 안내문 표시됩니다. <img alt="1 그림." src="/files/ftp_upload/3937/3937fig1.jpg" /> 1 그림. 분석의 원칙 (A)의 개요 및 방법 (B). A. 구체적으로 특정 poliovirus의 항원은 코발트 코팅 자성 구슬과 상호 작용하고 radioactively 레이블이 바이러스를 침전 것입 수있을 것입 인식 nanobodies을 그의 -이 태그로 지정했습니다. B. 그 .. 태그를 nanobodies는 radioactively 분류 poliovirus에 추가 incubated됩니다. 코발트 - 코팅 자석 구슬입니다추가 및 바운드 / 언바운드 항원의 자성 분리가 수행됩니다. 뜨는의 방사능이 측정되며 캡처한 항원의 양은 얻을 수 있습니다. <img alt="그림 2" src="/files/ftp_upload/3937/3937fig2.jpg" /> 그림 2. nanobody PVSS38C하여 다른 poliovirus의 항원의 마그네틱 비즈 친화 캡쳐. 표면에 뜨는에서 발견된 방사능의 비율은 PVSS38C의 농도의 함수로 표현된다. 14S의 경우 농도 - 반응 관계는 14S에 독점적으로 현재 에피토프로 PVSS38C의 상호 작용을 보여주는 찾을 수 있습니다. 아무도 상당한 상호 작용이없는 N-않으며이 아닌 특정 매우 높은 농도에서 캡처 무시할들이 언급되고 있지만, H 항원이, 감지가 가능하다. <img alt="그림 3" src="/files/ftp_upload/3937/3937fig3.jpg" /> 그림 3. nanobody Nb1하여 다른 poliovirus의 항원의 마그네틱 비즈 친화 캡쳐 </stronG&gt;. 표면에 뜨는에서 발견된 방사능의 비율은 Nb1의 농도의 함수로 표현된다. Nb1 어떤 poliovirus-항원 또는-subunit와 아무런 상호 작용이 없다 것으로 알려져 있습니다. 방사능의 100 %가 뜨는에서 발견되기 때문에 어떤 바이러스가 아닌 특별히 Nb1에 바인딩 없었다. 그림 2에 방사능의 손실 따라서 그 항원에 대한 nanobody의 구체적인 친화력에게만 때문입니다. <img alt="4 그림." src="/files/ftp_upload/3937/3937fig4.jpg" /> 4 그림. 분석의 재현성. 뜨는의 비율 방사능의 평균 가치는 nanobody의 농도의 함수로 표현된다. 실험은 다른 일에, 다른 사람에 의해 8 번 반복되었다. 표준 편차는 세로 막대로 표시됩니다.

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Chem. 76, 4175-4181 (2004).</li><li>Pelkmans, L., Kartenbeck, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caveolar+endocytosis+of+simian+virus+40+reveals+a+new+two-step+vesicular-transport+pathway+to+the+ER.">Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER.</a> Nat. Cell Biol. 3, 473-483 (2001).</li></ol>

우리는 공개 할게 없다.

프로토콜에서 nanobody 상호 작용 항원의 방사능이 뜨는에서 radiolabeled 바이러스의 손실로 정의됩니다. 따라서 시켰던 방사능 (= 100 - %)의 금액은 (자성 비즈에 바인딩) 뜨는 (= %)에서 방사능에 의해 간접적인 방법으로 추정됩니다. 한편 500 MM 이미다졸으로 자기 구슬에서 immunocomplexes를 eluting하여 직접적인 방법으로 항원의 시켰던 바운드 분수의 방사능을 측정하는 것도 가능합니다. 그것은 이전에 방...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong시약의&gt; 이름</strong</td&gt;<td<strong&gt; 회사 소개</strong</td&gt;<td<strong&gt; 카탈로그 번호</strong</td&gt;<td<strong&gt; 코멘트</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Dynabeads 그 .. 태그 분리 및 풀다운</td&gt;<td&gt; Invitrogen</td&gt;<td&gt; 101.03D</td&gt;<td&gt; 자성 구슬</td&gt;</tr&gt;<tr&gt;<td&gt; Optiphase &#39;HiSafe&#39;2</td&gt;<td&gt; Perkin 엘머</td&gt;<td&gt; 1200-436</td&gt;<td&gt; 섬광 유체</td&gt;</tr&gt;</tbody&gt;</table

a liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example

이 문서에서는 단순 양적, 액상 친화 캡쳐 분석이 제공됩니다. 한 단백질이 태그를 수 있도록 제공하여 다른 단백질이 라벨이 방법은 단백질 - 단백질 상호 작용 조사하기 위해 구현할 수 있습니다. 그것은 코발트 코팅 자성 구슬에 의해 태그가 단백질의 인식을하고 태그를 단백질과 레이블이 붙은 두 번째 특정 단백질 간의 상호 작용에있는 반면에 손을 대어를 기준으로합니다. 첫째, 라벨 및 태그 단백질을 섞어 실온에서 incubated됩니다. 태그를 인식 자석 구슬은, 추가하고 레이블이 단백질의 바운드 분율은 자석을 사용하여 언바운드 분획에서 분리됩니다. 캡처되는 분류 단백질의 양은 언바운드 분율에 남아 표시된 단백질의 신호를 측정하여 간접적 방법으로 결정될 수있다. conformational 변환 민감한 단백질 엉덩이가되었을 때 설명한 액체 위상 선호도 분석은 매우 유용합니다ayed. 분석의 개발 및 응용 프로그램은 poliovirus 및 nanobodies 1을 인식 poliovirus 간의 상호 작용에 대해 증명하고 있습니다. poliovirus는 단단한 표면 (미발표 결과)에 연결된 conformational 변환 2 ~ 민감한이므로 엘리사의 사용은 제한되며 액상 기반의 시스템은 따라서 선호한다. 자주 polioresearch 3,4에 사용되는 액상 기반 시스템의 예는 마이크로 단백질-immunoprecipitation 시험 5. 이 테스트는 적용을 입증하고있다하더라도, 그것은 6,7 nanobodies에 결석 FC-구조를 필요로합니다. 그러나 또 다른 기회로,이 흥미로운 안정 단일 도메인 항체 8은 쉽게 다른 태그로 조작 할 수 있습니다. 광범위하게 (그의) 6 태그와 같은 자신의 차례에 쉽게 자석 구슬에 코팅 수 니켈이나 코발트와 같은 이가 이온에 대한 친화력을 보여줍 사용됩니다. 따라서 우리는이 간단한 양적 개발친화 캡쳐 분석은 코발트 코팅 자석 구슬을 바탕으로. Poliovirus은 nanobodies과 unhindered 상호 작용을 가능하게하고 정량 검출이 가능하도록 35 S로 분류되었다. 방법은 수행이 용이하고 더 효과적으로 재생 자석 구슬의 가능성에 의해 지원되는 저렴한 비용으로 작성할 수도 있습니다.

Watch this Scientific Journal Video about A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example at JoVE.com

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

이 문서에서는 단순 양적, 액상 친화 캡쳐 분석이 제공됩니다. 그것은 한 손으로 그리고 다른 한편으로는 태그를 단백질과 두 번째 레이블이 붙은 단백질 (예 : poliovirus) 간의 친화력에 자석 구슬 및 태그 단백질 (예 : nanobodies) 사이의 상호 작용을 기반으로 안정적인 기술입니다.

단백질 - 단백질 상호 작용 연구를 자성 비즈를 사용하여 액상 동질 캡처 분석 : Poliovirus-Nanobody의 예

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

a-liquid-phase-affinity-capture-assay-using-magnetic-beads-to-study

Research

JoVE Journal

Biology

11.8K 조회수.  Vrije Universiteit Brussel. 이 문서에서는 단순 양적, 액상 친화 캡쳐 분석이 제공됩니다. 그것은 한 손으로 그리고 다른 한편으로는 태그를 단백질과 두 번째 레이블이 붙은 단백질 (예 : poliovirus) 간의 친화력에 자석 구슬 및 태그 단백질 (예 : nanobodies) 사이의 상호 작용을 기반으로 안정적인 기술입니다.

비디오: A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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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 ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

멀티 플렉스 항체 검사 방법을 사용하여 Luminex xMAP 분석에 캡처 엘리사의 전환

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

이분자 형광 보완의 유세포 분석 : 단백질 - 단백질 상호 작용을 연구하는 높은 처리량 정량 방법

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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 ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

IRE / IRP 예 : RNA - 단백질 상호 작용의 연구에 대한 전기 이동 기동성 교대 분석 실험 (EMSA)

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

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 ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.