Biz CF1β karşı antiserum için Olivier Vallon ederim. Bu çalışma Max Planck Topluluğu ve Deutsche Forschungsgemeinschaft (Schr 617/5-1) ve Bundesministerium für Bildung und Forschung (Sistem Biyolojisi Girişimi FORSYS, proje GoFORSYS) hibe tarafından desteklenmiştir.

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M., Muhlhaus, T., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+roles+of+molecular+chaperones+in+Chlamydomonas+and+Volvox.">New insights into the roles of molecular chaperones in Chlamydomonas and Volvox.</a> International review of cell and molecular biology. 285, 75-113 (2010).</li><li>Mayer, M. P., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperones:+cellular+functions+and+molecular+mechanism.">Hsp70 chaperones: cellular functions and molecular mechanism.</a> Cell Mol. Life Sci. 62, 670-684 (2005).</li><li>Schroda, M., Vallon, O., Whitelegge, J. P., Beck, C. F., Wollman, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chloroplastic+GrpE+homolog+of+Chlamydomonas:+two+isoforms+generated+by+differential+splicing.">The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing.</a> The Plant Cell. 13, 2823-2839 (2001).</li><li>Willmund, F., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEAT+SHOCK+PROTEIN+90C+is+a+bona+fide+Hsp90+that+interacts+with+plastidic+HSP70B+in+Chlamydomonas+reinhardtii.">HEAT SHOCK PROTEIN 90C is a bona fide Hsp90 that interacts with plastidic HSP70B in Chlamydomonas reinhardtii.</a> Plant Physiol. 138, 2310-2322 (2005).</li><li>Harris, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use. , (2008).</li><li>Mortensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSQuant,+an+open+source+platform+for+mass+spectrometry-based+quantitative+proteomics.">MSQuant, an open source platform for mass spectrometry-based quantitative proteomics.</a> J. Proteome Res. 9, 393-403 (2010).</li><li>M?hlhaus, T., Weiss, J., Hemme, D., Sommer, F., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Shotgun+Proteomics+Using+a+Uniform+15N-Labeled+Standard+to+Monitor+Proteome+Dynamics+in+Time+Course+Experiments+Reveals+New+Insights+into+the+Heat+Stress+Response+of+Chlamydomonas+reinhardtii.">Quantitative Shotgun Proteomics Using a Uniform 15N-Labeled Standard to Monitor Proteome Dynamics in Time Course Experiments Reveals New Insights into the Heat Stress Response of Chlamydomonas reinhardtii.</a> Mol. Cell Proteomics. 10, (2011).</li><li>Bukau, B., Horwich, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp70+and+Hsp60+chaperone+machines.">The Hsp70 and Hsp60 chaperone machines.</a> Cell. 92, 351-366 (1998).</li><li>Wandinger, S. 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Çıkar çatışması ilan etti.

Geçici protein-protein etkileşimleri (QUICK-X) yakalamak için bir çapraz adım ve eşitsiz çöktürme verimleri 6 normalleştirmek için bir kontrol Yağış: Biz son zamanlarda HIZLI anlayışın iki iyileştirmeler girmiştik. Burada HIZLI iki fazla iyileştirmeler içeren bir protokol mevcut: birincisi, biz 15 N metabolik etiketleme için SILAC 5 değiştirin. Avantajları 15 N basit inorganik tuz olarak sağlandığı takdirde 15 N metabolik etiketleme, SILAC daha ucuz olmasıdır. Bundan başka, 15 N metabolik etiketleme ile HIZLI birçok bitki, mantar ve bakteri gibi, tüm amino asitler için organizmalar Prototrofik için uygulanabilir. Ve nihayet, SILAC 5,6 doğasında arginin-to-prolin interconversion 15 N etiketli peptidler ölçümü için bir sorun teşkil etmiyor. 15 N proteomik verilerin nicel değerlendirmesi için uygun araçlar için örnekler MSQUANT 12 veya ben vardırOMIQS 13. İkinci olarak, özellikle bir karşı bir örnek olarak, belirli bir hedef proteinle etkileşime protein miktarını azaltmak için bir vasıta olarak yakınlık modülasyon tanıtmak. Bu yaklaşımın avantajı, bir model sistemlerini oluşturmak için zor olan ya da tüm proteinler, elzem hedef durumunda üretilen edilemez devirme mutant inşasında circumvents olduğunu vardır. Ayrıca, potansiyel bir hedef protein yikiyor için hücre bir tepki olarak ortaya çıkan diferansiyel protein ekspresyonu neden yanlış yorumlamaları önler: diğer proteinlerin yanı aşağı-regüle ve immünopresipitasyon için kullanılan antiserum ile çapraz reaksiyona, onlar yorumlanır hedef proteinin gerçek etkileşim ortakları olarak. Son olarak, afinite modülasyon uygun bir antikor-antijen ile-bulma oranı olan ihtiyacı ortadan kaldırmaktadır. Biz model organizma olarak Chlamydomonas reinhardtii bizim protokol uygulanmasına rağmen, Kolayca hücre kültürü içinde yetiştirilen ve azot kaynağı olarak amonyum nitrat ya da kullanabilir edilebilir herhangi bir diğer organizma için adapte edilebilir. ATP / ADP protein komplekslerinin Affinity modülasyon doğrudan doğruya GroEL/HSP60/Cpn60 ya da HSP90 şaperon sistemleri 14,15 gibi olan etkileşimi ve kohort substrat proteinleri ile ATP durumuna göre değişir diğer şaperonlar uygulanabilir, ya da herhangi bir diğer sistem ile yer edilebilir bağlanma afiniteleri ATP tarafından modüle edilir. Afinite modülasyon aynı zamanda protein etkileşimi ile afiniteleri HSP90 sistemleri 15 söz konusu olduğunda radicicol veya geldanamycin gibi belirli ilaç ile değiştirilmiş olduğu durumlarda için çalışması gerekir. Bizim protokol açık bir sınırlama gerektirir olmasıdır afiniteyle saflaştırılmış özel bir tedavi / ortağı proteinleri olan yakınlığından modüle ilaca duyarlı olduğu bilinen bir hedef proteine ​​karşı antikorlar. Bu nedenle, herhangi bir yüksek hacimli bir yöntemdir.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> ProteinA Sepharose </td><td> Sigma-Aldrich </td><td> P3391 </td><td></td></tr><tr><td> DMP (Dimetil pimelimidate) </td><td> Sigma-Aldrich </td><td> D8388 </td><td> -20 ° C&#39;de kurutulmuş Mağaza, kullanmadan önce doğrudan çözülür </td></tr><tr><td> (Tam, EDTA-ücretsiz) Proteaz inhibitörü </td><td> Roche Uygulamalı Bilimler </td><td> 11873580001 </td><td></td></tr><tr><td> ATP (adenozin-5&#39;-triphosphat) </td><td> Carl Roth </td><td> K054 </td><td></td></tr><tr><td> Kreatin fosfat </td><td> Sigma-Aldrich </td><td> 27920 </td><td></td></tr><tr><td> Kreatin fosfokinaz </td><td> Sigma-Aldrich </td><td> C7886 </td><td>; </td></tr><tr><td> DSP Dithiobis [süksinimidil propionat] </td><td> Sientific Thermo </td><td> 22585 </td><td> 4 at kurutulmuş Mağaza ° C </td></tr><tr><td> 15 NH4CI </td><td> Cambridge izotop laboratuvarları, Andover, MA </td><td> NLM-467 </td><td></td></tr><tr><td> Lys-C (Endoproteinase Lys-C) </td><td> Roche Uygulamalı Bilimler </td><td> 11047825001 </td><td></td></tr><tr><td> Tripsin boncuklar (Poroszyme Tripsin İmmobilize) </td><td> Applied Biosystems </td><td> 2-3127-00 </td><td> Doğrudan kullanmadan önce kuvvetlice karıştırın </td></tr><tr><td> Empore C 18 47 mm Disk </td><td> Varian </td><td> 12145004 </td><td></td></tr></tbody></table>

a protocol for the identification of protein-protein interactions based on 15n metabolic labeling, immunoprecipitation, quantitative mass spectrometry and affinity modulation

Protein-protein etkileşimleri hücrede birçok biyolojik süreçleri için temel vardır. Bu nedenle, karakterizasyonu araştırmalarını 1 mevcut olduğu için yöntemler mevcut araştırma ve bir bolluk önemli bir rol oynar. Protein-protein etkileşimleri genellikle son derece dinamik ve hücre içi lokalizasyonu, translasyon sonrası modifikasyonlar ve yerel proteini çevreye 2 bağlı olabilir. Bu nedenle, co-immünopresipitasyon yaklaşımlar tercih 3 yöntemi olduğu için, doğal ortamda araştırılması gerekmektedir. Co-Çökelen etkileşim ortakları bir hedeflenmemiş bir şekilde hedefe yönelik bir yaklaşım içinde, ya da kütle spektrofotometresi (LC-MS/MS) ile immünoblot yoluyla tespit edilir. Bu ikinci strateji genellikle olumsuz özellikle güvenli bir unspecifically tetikleyici proteinlerin izlerini tespit çağdaş kütle spektrometre ve yüksek hassasiyet elde edilen sahte pozitif buluşları, çok sayıda tarafından etkilenir. Bir recenBu sorunun üstesinden gelmek için t yaklaşım fikrine dayanır unspecifically tetikleyici protein miktarları etkilenmemiş olması gerekir, belli etkileşim ortak düşük miktarda hücresel konsantrasyon RNAi ile azaltılır, belirli bir hedef proteini ile ortak bir çökelti kalmayacaktır. Bu yaklaşım, Nakavt 4 ile birlikte kantitatif immünopresipitasyon QUICK adlandırılan yabani tip ve knock-down suşları immunoprecipitated protein miktarları ölçmek için Kararlı İzotop Hücre kültüründe Amino asitlerin Etiketleme (SILAC) 5 ve MS kullanır. 1:1 oranında bulunan kirletici proteinler, hedef proteinin özel etkileşim ortağı olarak yabani tip çökelti içinde zenginleştirilmiş olan olarak kabul edilebilir. Yenilikçi, HIZLI bazı kısıtlamalar taşır rağmen: İlk, SILAC maliyet-yoğun ve ideal arjinin ve / veya lizin için okzotrofik olan organizmalar için sınırlıdır. Addit Bunun yanında, arginin, ağır besleme durumunda, arginin-to-prolin interconversion sonuçional kitlesel bir peptid her prolin için vardiya ve biraz miktar daha sıkıcı ve 5,6 daha az hassas hale ışık arginin, ağır sulandırır. İkincisi, HIZLI antikorlar onlar knock-down mutantlar özler hedef proteini ile doymuş hale gelmez böyle titre gerektirir. Burada 15 N metabolik etiketleme için SILAC değiştirerek ve protein-protein etkileşimleri afinite modülasyonu için RNAi-aracılı knock-down değiştirerek HIZLI ve yukarıda belirtilen sınırlamaları aşan bir modifiye HIZLI protokol tanıtmak. Biz tek hücreli yeşil alg Chlamydomonas model organizma ve hedef protein 7 (Şekil 1) olarak kloroplast HSP70B eşlikçi olarak reinhardtii kullanarak bu protokolün uygulanabilirliğini göstermek. HSP70s özel ko-şaperonlar ve sadece ADP durum 8 yüzeyler ile etkileşim bilinmektedir. Bu özel kontrol etmek için bir araç olarak bu özellik faydalanmakonun nükleotid değişimi faktörü CGE1 9 ile HSP70B etkileşimi.

Watch this Scientific Journal Video about A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Aff

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Biz doğru ve yanlış protein-protein etkileşimleri ayırmak için önce tanıtıldı HIZLI (Nakavt ile birlikte kantitatif immünopresipitasyon) yaklaşımın bir çeşitleme sunuyor. Bizim yaklaşıma dayanmaktadır<sup&gt; 15N</sup&gt; Metabolik etiketleme, ATP, immünopresipitasyon ve kantitatif kütle spektrometresi varlığı / yokluğu ile protein-protein etkileşimleri yakınlıklarından modülasyonu.

Dayanarak protein-protein etkileşimleri ve kimliği için bir protokol<sup&gt; 15</sup&gt; K Metabolik Etiketleme, immünopresipitasyon, Kantitatif Kütle Spektrometrisi ve Affinity Modülasyon

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

20.4K Görüntüleme.  Max Planck Institute of Molecular Plant Physiology. Biz doğru ve yanlış protein-protein etkileşimleri ayırmak için önce tanıtıldı HIZLI (Nakavt ile birlikte kantitatif immünopresipitasyon) yaklaşımın bir çeşitleme sunuyor. Bizim yaklaşıma dayanmaktadır 15N Metabolik etiketleme, ATP, immünopresipitasyon ve kantitatif kütle spektrometresi varlığı / yokluğu ile protein-protein etkileşimleri yakınlıklarından modülasyonu.

Video: A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Numune Hazırlama: Ultra İnce Tabaka Yöntemi

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biyoloji

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Nicel Değerlendirme Elektrosprey İyonizasyon Kütle Spektrometresi kullanarak Maya Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Protein Kompleksi belirlenmesi<em&gt; Escherichia coli</em&gt; Tandem Kütle Spektrometresi ile Kombine Sıralı Peptid Affinity Arıtma kullanarak

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Multiprotein kompleksleri Karşılaştırmalı Analizi için Yeni Bir Yaklaşım dayanarak<sup&gt; 15</sup&gt; N Metabolik Etiketleme ve Kantitatif Kütle Spektrometre

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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.

Biyoparlaklık Rezonans Enerji Transferi kullanma Canlı Hücrelerde Protein-protein etkileşimleri araştıran

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

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Arabidopsis Protein-DNA Etkileşimleri Tespiti için Kromatin Immunoprecipitation Deneyi<em&gt; İn Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Tandem Affinity Arıtma kullanarak MyoD interaktom belirlenmesi kütle spektrometresi ile birleştiğinde

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

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.

Protein-protein etkileşimlerinin Autophagy araştırma çalışma

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

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Siteye özel Protein lizin asetilasyon ve Succinylation veri bağımsız edinme kütle spektrometresi kullanılarak Stoichiometry miktar

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Senoterapötik Ilaçların tanımlanması için SA-β-Galactosidaz tabanlı tarama tahlili

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...