AF는 유럽 공동체의 일곱 번째 프레임 워크 프로그램에서 유럽 연구위원회에서 시작 기금에 의해 지원되었다 (FP7 / 2007-2013) / ERC 부여 계약 N 203413을 ° 복잡한 systems.HA 바이오 하이브리드 및 AI에 대한 미네르바 센터가 지원됩니다 예루살렘의 히브리 대학에서 고급 학위 학생을위한 달리아 댄 Maydan 원정대에 의해.

1. 펩타이드 어레이 설계 <ol><li> 부분적으로 10 ~ 20 잔기 펩티드 겹치는에 표적 단백질의 서열을 나눈다. 특정 실험에 오버랩 량과 행하는 실험실 자원 있지만 원칙적 긴 오버랩을 더 다양하다. 펩타이드의 상호 작용에 대한 책임을 질 수있는 단백질 계정 알려진 이차 구조 요소를 고려 설계 할 때. </li><li> 상업 업체를 통해 설계 펩타이드 배열을 주문. 여기서, 사용 펩티드 공유 C 말단을 통해 어레이에 결합. </li></ol> 바인딩 2. 차단 비 특정 <ol><li> 0.05 % 트윈 -20 (TBST / PBST)를 함유하는 50mM 트리스 또는 포스페이트 완충액을 확인하고 (정확한 이온 강도를 알기 위해)의 HCl 또는 NaOH를 측정 된 양 및 NaCl을 사용하여 원하는 이온 강도와 원하는 pH로 조정 . 여기에서, 7.5의 pH 및 150 mm의 이온 세기를 사용한다. </li><li> 의 블로킹 용액을 만들기2.5 %는 (W / v)의 TBST / PBST에서 탈지유 분말. </li><li> 비특이적 결합을 방지하기 위해, 차단 용액 5ml에 배열 몰입. 2-4 상온에서 시간 또는 하룻​​밤 진탕 4 ºC에서의 배열을 품어. </li></ol> 3. 단백질과 배양 <ol><li> 실온에서 진탕 기에서 5 분 동안 5 ㎖를 TBST / PBST 회 30 초 동안 차단 용액과 5 ml의 제 배열을 씻는다. </li><li> (v가 ​​/ w) 비특이적 결합을 방지하기 탈지유 분말 2.5 %를 함유하는 그의 태그 된 단백질 용액 5 ㎖로 세척하고 어레이를 부화. 여기서, 설명 블로킹 용액에 용해 된 단백질 용액의 4.5 μM (STIL 500-650)을 사용한다. NOTE : 보통 5-10 μM 단백질은 선별을 위해 사용되지만, 단백질 농도는 결합 친화력과의 로컬 어레이에 결합 된 펩타이드의 농도 (효율에 따라, 심지어 낮은 2-3 μM 일 수있다 합성). 버퍼있는 단백질다양 할 수 있지만, 상술 한 용해 같은 차단 용액을 사용하여 단백질을 희석하는 것이 일반적이다. </li><li> 실온에서 밤새 또는 4 ºC에서 3-8 시간 동안 단백질 용액을 인큐베이션 어레이. </li></ol> 4. 항체와 함께 배양 <ol><li> 5 ml의 TBST / PBST로 배열을 세 번 씻으십시오. 처음 세탁은 실온에서 진탕에 두 5 분 세척 한 다음 30 초입니다. </li><li> 실온에서 쉐이커상에서 1 시간 동안 블로킹 용액과 동일한 농도에서 동일한 성분을 함유 배양 완충액 : 희석 된 HRP 컨쥬 게이트 된 항체 5 ㎖ (1500 1)로 세정 배열 부화. 항체는 표적 단백질 또는 태그 중 하나를 결합 할 수있다. </li><li> 인트없이 동일한 프로토콜을 반복함으로써 배열 표적 단백질 (예를 들어, 올리고머 화를위한 연구)로부터 펩티드를 포함 특히 어레이 펩타이드 항체의 상호 작용을 시험 대조 실험을 수행P (3), 단백질과 인큐베이션. </li><li> 5 ml의 TBST / PBST로 배열을 세 번 씻으십시오. 처음 세탁은 실온에서 진탕에 두 5 분 세척 한 다음 30 초입니다. </li></ol> 5. 배열을 읽기 <ol><li> ECL 웨스턴 블로 팅 기판 키트와 함께 발광 개발을 수행하십시오. </li><li> 발광 이미지 분석기로 검출을 수행. </li><li> 결과를 분석합니다. 어레이의 두 중복이 같은 신호를 보여, 그래서 결과를 신뢰할 수 있는지 확인합니다. 파트너 단백질의 구조가 알려지면, 결합 펩티드 구조에서 검색하고 결합 부위를 찾는다. 순서에 멀리있다하더라도 펩타이드는 차 구조에 가깝게하고 구속력있는 사이트를 만들 수 있습니다. </li></ol>

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The authors have nothing to disclose.

펩타이드 어레이 스크리닝 파트너에서 표적 단백질의 결합 부위를 식별하기위한 훌륭한 도구이다. 상기 분석을 수행하기 쉽고 빠르게 결과는 하나 또는 두 개의 일 얻을 수있다. 펩타이드 어레이 스크리닝 다재다능 많은 목적을 위해 사용될 수있다. 그것은 예컨대 인산화와 같은 번역 후 변형을 특성화하고 키나제의 기판을 식별하는데 사용될 수있다. 예 : 급성 골수성 백혈병 관련이 FLT3 키나제,...

단백질 - 단백질 상호 작용은 살아있는 세포에서의 처리의 대부분을 매개한다. 장애인 단백질 상호 작용은 단백질 상호 작용에게 중요한 약물 표적을, 질병의 원인이 될 수 있습니다. 이것은 분자 레벨에서의 이러한 상호 작용을 이해하는 것이 매우 중요하다. 단백질 상호 작용은 세포의 생화학 적 분석에서 양적 생물 리 학적 분석법에 이르는 다양한 기술을 이용하여 연구하고 있으며, 이들 도메인은 단백질 또는 펩타이드와, 전체 길이 단백질과 중 수행 될 수있다. 펩타이드는 단백질 상호 작용을 연구하는 우수한 도구를 제공합니다. 펩티드는 쉽게 합성 한 손과 반면 1,2에 고 스루풋 방식으로 다수의 표적 단백질에 특이 적 상호 작용 부위에 초점을 허용 할 수 있기 때문이다. 펩타이드 어레이 스크리닝은 빠르고, 단시간 (3)에 다수의 파트너와 표적 단백질의 상호 작용에 관한 많은 양의 데이터를 획득하기위한 방법을 수행하기 쉬운. 단백질 - 단백질 상호 작용을 검출하고 분석하기위한 다른 생화학 또는 생물 물리학 적 방법과 달리, 펩티드 배열 선별은 단백질의 매우 낮은 농도를 필요로하며, 매우 약한 결합 검출 할 수있다. 펩타이드 배열이 효소의 활동 (6)과 높은 처리량을 연구, 단백질 - 단백질 또는 수용체 - 리간드 상호 작용 사이트 4, 동종 또는 이종 올리고머 인터페이스의 매핑과 같은 펩타이드 - 단백질 상호 작용에 많은 응용 프로그램의 특성 항체를 5 항원 사용할 수있는 구조 - 활동 관계 (SAR)는 7 연구. 펩타이드 배열 검사에 대한 심도있는 검토를 위해 카츠 등을 참조하십시오. 4 펩타이드 어레이의 여러 유형 현재 존재한다. 펩타이드의 합성을 주로 SPOT 기법 4,8를 사용하여, 직접 고체 지지체에 펩티드의 고체 지지체, 또는 합성을 부착하기 전에 : 펩타이드 어레이를 만들기위한 두 가지 주요 합성 전략이있다.펩티드는 9- 플루 오레 닐 옥시 카보 닐 (Fmoc) 화학 (8)에 의해 일반적으로 고체 지지체 상에 합성된다. 일반적인 합성 기법 중 C 말단을 통해 펩타이드 N 말단을 통해 첨부 파일 (예를 들어, JPT의 pepstar 배열 9) 및 펩타이드 첨부 파일은 (예를 들어, PEPSCAN의 pepchip 어레이 (10), JPT의 pepspot 배열 9 INTAVIS의 celluspot 어레이 (11)) 4. 고체 지지체는 다양하며, 그래서 그것에 펩타이드 결합의 화학적 성질을 수행 할 수 있습니다. Cys 또는 말단 펩티드는 티올 기 (12)를 통해 유리 슬라이드에 부착 될 수있다. 펩티드의 N 말단이 공유 결합 된 아미노산의 에스테르 화를 통해 셀룰로오스 막에 수산기에 결합 될 수있는 8 첨부. 여기서 우리는 단백질 - 단백질 상호 작용을 연구하기위한 방법으로 펩타이드 어레이 스크리닝 상세한 프로토콜을 제시한다. 우리가 사용하는 배열은 큰 AMO를 포함하는 마이크로 어레이 인 Celluspots 배열입니다유리 슬라이드에서 지원하는 작은 셀룰로오스 막에 점 (384 점 중복)의 UNT. 이 낮은 단백질의 볼륨과 항체 작업과 하나의 실험 당 데이터의 상당한 양을 획득 할 수 있습니다. 이 배열은 또한 바인딩 선호도가 낮은 검출 할 수 있습니다 높은 펩타이드 밀도가 포함되어 있습니다. 어레이는 정상 세포 증식 (13)을 제어하기에 매우 중요하다 STIL-CHFR 상호 작용을 매핑하기 위해 사용되었다. 두 단백질 사이의 상호 작용은 제어되지 않은 암의 발생을 초래할 수있다. 이 상호 작용을 매핑하여 우리는 특정 결합 부위와 잔여 14 바인딩을 발견했다. 이것은이 단백질 - 단백질 상호 작용을 억제하는 억제제 합리적 설계를위한 길을 열어.

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:231px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptide array</td>
			<td>INTAVIS Bioanalytical Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">His-probe Antibody (H-3) HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8036 HRP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-ECL Kit</td>
			<td>Biological industries</td>
			<td>20-500-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skim Milk</td>
			<td>Becton, Dickinson and Company</td>
			<td style="width:119px;">232100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LAS-3000 camera&#160;</td>
			<td>FUJI film</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction sites using peptide arrays

단백질 - 단백질 상호 작용은 유기체의 살아있는 세포의 항상성 및 제어 공정의 대부분을 매개한다. 장애인 단백질 상호 작용은 단백질 상호 작용에게 중요한 약물 표적을, 질병의 원인이 될 수 있습니다. 이것은 분자 레벨에서의 이러한 상호 작용을 이해하는 것이 매우 중요하다. 단백질 상호 작용은 세포의 생화학 적 분석에서 양적 생물 리 학적 분석법에 이르는 다양한 기술을 이용하여 연구하고 있으며, 이들 도메인은 단백질 또는 펩타이드와, 전체 길이 단백질과 중 수행 될 수있다. 펩타이드는 펩타이드가 용이하게 합성 할 수 있기 때문에 단백질의 상호 작용을 연구하고, 특정 상호 작용 사이트에 집중 할 수 있도록 우수한 도구를 제공합니다. 펩타이드 어레이는 치료 목적 표적 단백질에 결합 펩티드 두 단백질 사이의 상호 작용 위치의 식별뿐만 아니라 스크리닝을 가능하게한다. 그들은 또한 높은 처리량 SAR 연구를 할 수 있습니다. 결합 부위, typi의 식별을위한학적 펩타이드 어레이는 일반적으로 부분적으로 원하는 타겟 단백질의 하나 또는 그 이상의 파트너 단백질의 전체 서열로부터 유도 된 10 내지 20 잔기 펩티드 겹치는 포함. 표적 단백질을 바인딩 어레이 스크리닝 단백질 만 소량 사용 쉽고 빠른 방법에서 파트너 단백질의 결합 부위에 대응하는, 펩티드 결합을 보여준다. 이 문서에서 우리는 목적 단백질과 파트너 사이의 상호 작용 사이트를 매핑하는 펩타이드 배열을 선별하기위한 프로토콜을 설명합니다. 펩티드 배열은 고려 그들의 이차 구조를 고려 파트너 단백질의 서열에 기초하여 설계된다. 이 프로토콜에 사용되는 배열은 INTAVIS 바이오 어낼 리티 컬 인스트루먼트 준비 Celluspots 배열했다. 이 배열은 바인딩 불특정 한 후 연구 단백질 배양 방지하기 위해 차단됩니다. 항체를 이용하여 검출 단백질 간의 특이 적 상호 작용 부위에 대응하는 펩타이드 결합을 보여준다.

STIL은 매우 중요 centrosomal 단백질이다. 19 - 그것은 정상적인 세포 분열 및 세포 증식을 13, 15를 제어합니다. STIL 여러 단백질 18,20,21과 상호 작용하고 상호 작용의 대부분은 본질적으로 무질서 지역 (IDR) (14) 자사의 중앙 부분을 통해 발생합니다. 우리는 STIL 결합 단백질 CHFR에서 유래 펩타이드로 구성된 배열을 설계했습니다. CHFR 스트레스 22 유사 분열에 응답...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction Sites Using Peptide Arrays at JoVE.com

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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 ...

identifying-protein-protein-interaction-sites-using-peptide-arrays

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17.9K 조회수.  The Hebrew University of Jerusalem. 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.

비디오: Identifying Protein-protein Interaction Sites Using Peptide Arrays

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

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

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

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

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

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

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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 ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

이미징 단백질 - 단백질 상호 작용 생체내에</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

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 ...

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. ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

SPOT 펩타이드 배열을 사용하여 단백질 리신 Methyltransferases의 특이성 분석

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

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 ...