재생 의료 보조금 RS1 - 00331-1 캘리포니아 연구소 RL1 - 00682-1 (Z. 장에), 미국 파킨슨병이 작품은 NIH 보조금 RO1 DC006497, RO1 NS057289하고, PO1 ES016738 (Z. 장)를 지원했습니다 질병 협회 (Z. 장 및 YS 츄)을, 그리고 파킨슨병 연구에 대한 마이클 J. 폭스 재단 (Z. 장).

교양 세포에서 단백질의 ubiquitination 검색 <ol><li> 관심 단백질과 (에피토프 - 태그의 버전) ubiquitin을 표현 plasmids와 Transfect 교양 세포. </li><li> 전체 세포 용해 완충액 (2 % SDS, 150 MM NaCl, 10 MM 트리스 - HCL, pH를 8.0) 2mM 나트륨 orthovanadate, 50 MM 나트륨 불화, 그리고 프로 테아제 억제제와 함께.하십시오 </li><li> 접시 당 100 μl 세포 용해 완충액 (6cm 요리)와 세포를 Lyse. 큰 접시를 사용하는 경우, 적절하게 볼륨을 조절할 수 있습니다. 소용돌이는 음식을 신중하게 용해 버퍼가 성장 세포의 전체 영역을 커버하도록. </li><li> 휴대 스크레이퍼로 세포를 수집하고 1.5 ML eppendorf 튜브에 세포 lysates를 전송할 수 있습니다. 10 분 끓인 즉시 뜨거운 접시에 튜브를 놓습니다. </li><li> sonication 장치와 전단은 세포. </li><li> 희석 버퍼의 900ul (10 MM 트리스 - HCL, pH를 8.0, 150 MM NaCl, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 1 %의 트리톤) 추가합니다. 회전 30-60 분 4 ° C에서 샘플을 품어. </li><li> 30 분 20,000 XG에서 희석 샘플을 스핀. 새로운 eppendorf 튜브에 뜨는 결과를 전송합니다. 펠렛을 방해하지 않도록주의하십시오. </li><li> 단백질 농도를 측정. </li><li> 호환 버퍼 (50 % 슬러리)의 목표 단백질에 대한 단백질 또는 G - 아가로 오스 비드 - 복합 항체를 준비합니다. P - 200 피펫 팁의 좁은 끝을 잘라 immunoprecipitation을위한 준비 세포 lysates의 500-1,500 μg에 수지 14-20 μl를 전송합니다. 높은 transfection 효율과 전지, 500 μg 충분합니다. 낮은 transfection 효율 세포에 대한 더 많은 단백질이 필요합니다. </li><li> ° C 하룻밤 회전 4 셀 lysate - 비드 혼합물을 품어. </li><li> 5 분 5,000 XG에 비즈를 스핀 다운. 뜨는을 대기음. 세탁 버퍼 (10 MM 트리스 - HCL, pH를 8.0, 1 M NaCl, 1 MM EDTA (에틸렌 다이아 민 테트라 초산), 1 % NP - 40)가 두 번있는 수지를 씻으십시오. </li><li> 30 초에 대한 20,000 XG에서 마지막 시간 동안 비즈를 스핀. 잔여 세척 버퍼를 기음과 2X SDS 로딩 버퍼와 수지를 끓여. </li><li> 분석 immunoblotting에 대한 SDS - PAGE 젤로 샘플을로드합니다. </li><li> 각각의 항체와 ubiquitin과 대상 단백질 감지. immunoblotting을 위해, 우리는 일반적으로 첫번째 ubiquitin 감지합니다. 막은 다음 단백질 시켰던를 감지하는 데 사용됩니다. </li></ol> 체외의 ubiquitination 분석을 통해 대상 단백질의 ubiquitination의 감지 <ol><li> 배 ubiquitination 버퍼를 (100 MM 트리스 - HCL, pH를 7.5, 25 MM MgCl2, 2.5 MM DTT, 10 MM ATP)합니다. 최대 6 개월에 대해 -20 ° C에서 작은 aliquots에 저장합니다. 일부 프로토콜은 또한 ATP 재생 1,2에 대한 버퍼로 크레아틴 인산염과 크레아틴 키나제를 추가합니다. </li><li> 각 반응은 다음을 포함하는 혼합물을 준비 : <table border="1"><tbody><tr><td> μl 8 </td><td> 배 ubiquitination 버퍼 </td></tr><tr><td> 250 NG </td><td> ubiquitination E1 </td></tr><tr><td> 500 NG </td><td> ubiquitination E2 </td></tr><tr><td> 0.5 μg </td><td> ubiquitin </td></tr><tr><td> 0.5 μg </td><td> 관심 단백질 </td></tr><tr><td></td><td> 40 μl 전체 볼륨을 물 </td></tr></tbody></table> 컨트롤로, E1, E2, 또는 ubiquitin 중의 부재에서 유사한 반응을 준비합니다. </li><li> ° C 1시간 이상 37 혼합물을 품어. </li><li> SDS - PAGE 샘플 버퍼를 추가하여 반응을 중지하고 10 분에 대한 샘플을 끓여. </li><li> 분석 immunoblotting에 대한 SDS - PAGE 젤로 샘플을로드합니다. </li><li> 각각의 항체와 ubiquitin과 대상 단백질 감지. </li></ol>

<ol>
	<li>Xiong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1,+and+DJ-1+form+a+ubiquitin+E3+ligase+complex+promoting+unfolded+protein+degradation.">PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation.</a> J Clin Invest. 119, 650-660 (2009).</li><li>Xirodimas, D. P., Saville, M. K., Bourdon, J. C., Hay, R. T., Lane, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdm2-mediated+NEDD8+conjugation+of+p53+inhibits+its+transcriptional+activity.">Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.</a> Cell. 118, 83-97 (2004).</li><li>Didier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF5,+a+RING+finger+protein+that+regulates+cell+motility+by+targeting+paxillin+ubiquitination+and+altered+localization.">RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization.</a> Mol Cell Biol. 23, 5331-5345 (2003).</li><li>Laney, J. D., Hochsetrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+14.5+Analysis+of+Protein+Ubiquitination.">Unit 14.5 Analysis of Protein Ubiquitination.</a> Current Protocols in Protein Science. , 14.5.1-14.5.11 (2002).</li></ol>

The authors have nothing to disclose.

이 프레 젠 테이션에서, 우리가 처음 교양 포유 동물 세포에 대한 관심의 단백질에 ubiquitin 수정을 감지 허가 단계를 설명했다. 하지 않은 covalently 상호 작용하는 단백질에 대한 관심의 단백질에 특별히 ubiquitination를 감지하기 위해, 우리는 세포 용해, immunoprecipitation과 1,3 세척을위한 엄격한 조건을 사용합니다. 안티 ubiquitin immunoblotting 다음과 같은 거친 상태로 표적 단백질의 immunoprecipitation 감지 Ubiquitination은, 따라서 대상 단백질에 구체적으로 가능성이 높습니다. 실험 절차 중에 단백질 deubiquitination을 방지하기 위해, 같은 N - ethylmaleimide과 ubiquitin 알데히드와 같은 deubiquitinating 효소 억제제는 모든 버퍼 4 추가할 수 있습니다. 그러나, 그것은 deubiquitination 효소는 10 분 끓는 절차 후에 활성 상태로 유지 않을 수도 있습니다. 높은 분자 종의 강력한 얼룩이나 사다리 일반적으로 ubiquitination의 결과입니다. 관심의 단백질의 ubiquitination의 정도는 여러 가지 실험 조건에서 ubiquitinated / 수정되지 않은 대상 단백질의 비율을 비교하여 평가하실 수 있습니다. 대상 단백질의 ubiquitination이 증가되었을 때 한편, 무료 ubiquitin 분자는 일반적으로 감소합니다. 이 프로토콜은 또한 sumolyation 및 neddylation 같은 다른 ubiquitin과 같은 작은 분자 수정을 감지하는 데 유용합니다. 우리는 또한 정화 또는 재조합 단백질을 사용하여 체외의 ubiquitination 분석에서 설명했다. 다양한 에피토프 - 태그 ubiquitination 구성 요소는 상업적으로 사용할 수 있습니다. Ubiquitination의 E3 ligase의가 체외에서 ubiquitin 활용을 위해 필요하지 않습니다.

detection of protein ubiquitination

Ubiquitination, 단백질을 타겟으로 polypeptide ubiquitin의 공유 결합 첨부 파일 세 효소의 집합으로 실시하는 핵심 posttranslational 수정합니다. 그들은 ubiquitin - 활성화 효소 E1, ubiquitin - 결합 효소 E2, 그리고 ubiquitin ligase의 E3를 포함합니다. E1과 E2, E3 ubiquitin ligases 디스플레이 기판 특이성을 달리. 한편, 수많은 deubiquitylating 효소는 polyubiquitinated 단백질을 처리하는 역할을합니다. Ubiquitination 단백질의 안정성, 휴대 현지화, 그리고 생물 학적 활동의 변화가 발생할 수 있습니다. ubiquitination / deubiquitination 경로 또는 변경 ubiquitin 시스템 기능에 관련된 유전자의 돌연변이 같은 암, neurodegeneration, 그리고 신진 대사 장애의 다양한 유형으로 다양한 인간 질환과 관련된 있습니다. 대상 단백질의 변경 또는 정상 ubiquitination의 감지는 이러한 질병의 pathogenesis에 대한 더 나은 이해를 제공할 수 있습니다. 여기, 우리는 교양 세포에 단백질 ubiquitination을 감지 프로토콜 설명<em&gt; 생체내에</em&gt; 테스트 튜브<em&gt; 체외에서</em&gt;. 이 프로토콜은 또한 sumolyation 및 neddylation 같은 다른 ubiquitin과 같은 작은 분자 수정을 감지하는 데 유용합니다.

Watch this Scientific Journal Video about Detection of Protein Ubiquitination at JoVE.com

Detection of Protein Ubiquitination

Ubiquitination 세 효소의 집합으로 실시하는 핵심 posttranslational 수정합니다. 이 수정에 관여 유전자의 돌연변이가 여러 가지 인간의 질병와 관련된 있습니다. 여기, 우리는 교양 세포에 단백질 ubiquitination을 감지 프로토콜 설명<em&gt; 생체내에</em&gt; 테스트 튜브<em&gt; 체외에서</em&gt;.

단백질 Ubiquitination의 감지

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Nanyang Technological University

Research

JoVE Journal

Biology

42.7K 조회수.  The Sanford Burnham Institute for Medical Research. Ubiquitination 세 효소의 집합으로 실시하는 핵심 posttranslational 수정합니다. 이 수정에 관여 유전자의 돌연변이가 여러 가지 인간의 질병와 관련된 있습니다. 여기, 우리는 교양 세포에 단백질 ubiquitination을 감지 프로토콜 설명 생체내에 테스트 튜브 체외에서.

비디오: Detection of Protein Ubiquitination

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

전체 몽키 브레인에서 대규모 단백질 탐지에 대한 배치 Immunostaining

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

연구

생물학

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

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

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

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

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

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 ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

마이크로 패턴의 표면에 단백질 - 단백질 상호 작용에 - 생체내 감지

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

게이트웨이 가능 Bimolecular의 형광 Complementation (BiFC) 시스템을 사용하여 공장에서 단백질 상호 작용의 검출

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Sumoylation과 신진 효모 동원체 단백질 Ndc10과 Ndc80의 유비퀴틴 화를 감지 할 수 있습니다 단백질 정제 기술

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

공 초점 현미경으로 리간드 - 활성화 된 G 단백질 결합 수용체 내재화 검출

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

단백질 단백질 상호 작용 Autophagy 연구에서의 연구

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...