Die Arbeit in unserem Labor wird durch Zuschüsse von NIH, USDA National Institute of Food and Agriculture, NSF, BARD, DOE und BSF zu VC unterstützt

1. Expression und Extraktion der Proteine <ol><li> Unterschiedlich tag die Proteine ​​für ihre Erkennung getestet werden. Beschriften Sie die Protein auf der Membran (ProIM) mit einem Tag einer größeren Größe (zB GST) immobilisiert werden und die nicht kondensierten Tag kann als ein immobilisiertes negative Kontrolle (ProIMnc) verwendet werden. Beschriften Sie die Protein als löslich Sonde (PROSOL) entweder mit einem großen oder einem kleinen Etikett verwendet werden. Sicherung der gleichen Tag, um eine negative Kontrolle lösliches Protein (ProSOLnc) und beinhalten in der Interaktion Test, um die Spezifität der nachgewiesenen Bindung zu bestätigen. </li><li> Wählen Sie die Protein-Expression System tagged Proteine ​​zu exprimieren, dh E. coli, Baculovirus, etc., je nach Anforderung für potenzielle posttranslationalen Modifikationen und Renditen der Proteine. </li><li> Auszug ProIM und ProIM nc aus dem Organismus der Wahl (Schritt 1,2) mit SDS-PAGE-Ladepuffer (20% Glycerin, 4% SDS, 20 mM Tris-HCl, pH 6,8) mit Proteinase-Inhibitor-Cocktail. Verlassen Sie die Zellsuspension bei Raumtemperatur für 15 min, mit 0,5% 2-Mercaptoethanol und kochen für 5 min. </li><li> Auszug PROSOL und PROSOL nc aus dem Organismus der Wahl (Schritt 1,2) mit einem Standard-Protokolle 16. Diese Proteine ​​werden sollte leicht löslich in Bindungspuffer (140 mM NaCl, 10 mM, Tris-HCl pH 7,4, 2 mM EDTA, 2 mM DTT, 1% BSA, 0,1% Tween 20) verwendet in den Schritt 4.1. </li><li> Bestimmen Sie die Konzentration der rekombinanten Proteine ​​mit einem Standard-Verfahren, wie zB Bio-Rad Protein Assay Kit. wie Bio-Rad Protein Assay Kit. Wenn Rohextrakt, anstatt gereinigtes Protein, für den Assay verwendet wird, schätzen die Konzentration des Proteins von Interesse in diesem Extrakt durch Scannen Densitometrie der entsprechenden Band auf einem SDS-Polyacrylamidgel mit Coomassie Brilliant Blue R-250 und mit bekannten Konzentrationen von BSA als Referenz. </li></ol> 2. Immobilisierung von ProIM und ProIMnc auf der Membran <ol><li> Resolve zwei Sätze von Extrakten, die jeweils 1 pg ProIM und ProIM nc auf einem SDS-Polyacrylamidgel nach dem Standardprotokoll 16. </li><li> Nach der Elektrophorese, platzieren Sie den Transfer des Gels in 100 ml Transferpuffer (60 mM Glycin, 10 mM Tris, 0,0006% SDS, 20% MeOH) und inkubieren unter leichtem Schütteln für 20 min bei Raumtemperatur. </li><li> Elektrotransfer die Proteine ​​in dem Gel auf eine Nitrozellulose-Membran enthielt nach dem Standardprotokoll 16. </li></ol> 3. Re-Faltung von Membran-gebundenen Proteine <ol><li> Nach Elektrotransfer, inkubieren Sie die Membran für 15 min in 15 ml Puffer A (30 mM Tris-HCl pH 7,4, 0,05% Tween 20) unter leichtem Schütteln, um restliches SDS zu entfernen. </li><li> Nach dem Ablassen sorgfältig, übertragen Sie die Membran in 25 ml Denaturierungspuffer (7 M Guanidin-Hydrochlorid, 2 mM EDTA, 50 mM DTT, 50 mM Tris-HCl pH 8,3). und für 2 Stunden bei Raumtemperatur unter leichtem Schütteln inkubieren. (Hinweis: Die Nitrozellulosemembran wird undurchsichtig, wenn in der Denaturierung Puffer). und für 2 Stunden bei Raumtemperatur unter leichtem Schütteln inkubieren. </li><li> Übertragen Sie die Membran in 25 ml TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7,4) und Inkubation unter leichtem Schütteln für 5 min (Anmerkung: In diesem Schritt gewinnt die Membran ihre ursprüngliche weiße Farbe). </li><li> Übertragen Sie die Membran in 25 ml Bindungspuffer (siehe Schritt 1.2) und inkubieren Sie bei 4 ° C unter leichtem Schütteln über Nacht. </li></ol> 4. Probing the ProIM durch PROSOL <ol><li> Schneiden Sie die Membran in zwei Streifen, die beide mit ProIM und ProIM nc. </li><li> Machen Sie den PROSOL und ProSOLnc Hybridisierung durch Verdünnung der Vorbereitung mit 1-10 ug entweder PROSOL oder ProSOLnc in 10 ml frisches Bindungspuffer. Übertragen Sie jede Membran in die PROSOL oder ProSOLnc Hybridisierungslösung und Inkubation für 1,5 h bei Raumtemperatur unter leichtem Schütteln. </li><li> Entfernen Sie die Membranen, die aus der Hybridisierung Lösungen und spülen dreimal für je 15 min in TBS. </li></ol> 5. Visualisierung von Protein-Protein-Interaktion durch Immunoblot <ol><li> Blockieren Sie die Membran mit 2,5% Magermilch in TBST (10 mM Tris-HCl, 140 mM NaCl, 0,05% Tween 20, pH 7,4) für 1 h bei Raumtemperatur. Verdünnen Sie die primären Antikörper (Anti-strepII polyklonale Kaninchen-Antikörper) in 0,5% Magermilch in TBST bei der Konzentration der vom Hersteller empfohlen. </li><li> Legen Sie die verstopfte Membranen in der Antikörper-Lösung, und für 1 h bei Raumtemperatur oder über Nacht bei 4 ° C unter leichtem Schütteln. </li><li> Spülen Sie die Membranen in 20 ml TBST für 15 min und zweimal für 5 min bei Raumtemperatur unter leichtem Schütteln. </li><li> Verdünnen Sie die Sekundär-Antikörper (Anti-Kaninchen-IgG-Antikörper) konjugiert mit Meerrettichperoxidase (HRP) in 0,5% Magermilch in TBST bei der Konzentration der vom Hersteller empfohlen. Legen Sie die Membranen in der Sekundär-AntikörperLösung und Inkubation für 1 h bei Raumtemperatur unter leichtem Schütteln. </li><li> Spülen Sie die Membranen in 20 ml TBST für 15 min und zweimal für 5 min bei Raumtemperatur unter leichtem Schütteln. Nach dem letzten in TBS spülen, visualisieren die Protein-Protein Interaktion mit einem HRP Chemilumineszenz Substrat (z. B. Millipore Immobilon westlichen Chemilumineszenz HRP Substrat). </li><li> Probe der gleichen Membranen mit dem primären Antikörper für das Tag fusioniert ProIM, durch die entsprechenden Sekundär-Antikörper wie in den Schritten von 5,1 bis 5,4 beschrieben wird. Visualisieren Sie ProIM und ProIMnc wie beschrieben in Schritt 5,5 bis die Identität des Bands in die Proteinmembran Overlay-Assay (Schritt 5,5) erhalten zu validieren. In den meisten Fällen erfolgt das Ausbrechen nicht notwendig, da das Signal von diesem Schritt erhalten ist viel stärker als Restsignal von Schritt 5,5. </li></ol> 6. Repräsentative Ergebnisse: Die ANK-MP-Interaktion wurde durch BIFC in Tabak Epidermiszellen (Abbildung 1A) beobachtet. Da MP ist ein hoch unlösliche Proteinablagerungen, wenn in Bakterien oder in Pflanzen exprimiert, wurde das Protein-Membran-Overlay-Assay angenommen, um diese Interaktion in vitro (Abbildung 1B) zu validieren. Proteinextrakte mit 1 pg GST-MP (ProIM) oder ungesichert GST (ProIM nc) wurden durch SDS-Polyacrylamidgelelektrophorese von Elektrotransfer auf eine Nitrozellulosemembran folgte aufgelöst. Wenn diese ProIMs mit löslichen ANK-strepII (PROSOL), GST-MP, aber nicht fusionierten GST wurden sondiert, zeigten Bindung (Abbildung 1B, Bahnen 1, 2, auf den Spuren 5, 6 zu vergleichen). Außerdem, wenn die gleiche Menge von ProIMs mit einem unabhängigen, ProSOLnc, dh Arabidopsis zytoplasmatischen NADH-Kinase markiert strepII (NADH3-strepII), keine Bindung wurde nicht beobachtet wurden sondiert ein weiterer Beweis für die Spezifität der ANK-MP-Wechselwirkung (Abbildung 1B, Bahnen 3, 4). <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Spezifische Bindung von Tabak ANK zu TMV MP in vivo und in vitro. (A) ANK-MP-Interaktion in lebenden Tabak Epidermiszellen wie BIFC erkannt. Starke YFP-Signal rekonstruiert wurde, wenn MP und ANK, um C-terminal und N-terminalen Hälften von YFP bzw. verschmolzen, wurden in Tabak Epidermis folgenden microbombardment ihrer kodierenden Gene koexprimiert. Diese BIFC Signal in puncta an der Zellperipherie, die Diagnose von Plasmodesmen 15 sind angesammelt. (B) ANK-MP-Interaktion in vitro durch Protein-Membran-Overlay-Assay nachgewiesen. Proteinextrakte mit 1 pg GST-MP (ProIM) oder ungesichert GST (ProIMnc) wurden auf einem 15% SDS-Polyacrylamidgel von Elektrotransfer auf eine Nitrozellulosemembran folgte aufgelöst. Die GST-MPProIM und GSTProIMnc wurden mit 0,5 ug / ml ANK-strepII (PROSOL) inkubiert und ANK Bindung wurde durch Untersuchung der Membran mit anti-strepII polyklonalen Kaninchen-Antikörper, gefolgt von Anti-Kaninchen-IgG + M konjugierten Sekundärantikörper detektiert HRP (Spuren 1 und 2). Weder GST-MPProIM noch GSTProIMnc mit einem nicht verwandten Proteinen, Arabidopsis zytoplasmatischen NADH-Kinase, fusioniert mit dem strepII tag (ProSOLnc, Bahnen 3 und 4) interagiert. Die Identität der Band in diesem Assay beobachtet wurde durch Untersuchung der Membran mit anti-GST-Antikörper (Spuren 5 und 6) bestätigt. Wenn die Membran mit Denaturierungspuffer wurde, ohne mit Puffer A gewaschen behandelt werden, ist die Bindung des GST-MP zu ANK verloren, während nicht identifizierte Proteine ​​in der GST-MP und GST contining Eiweißextrakte enthaltenen reagierte mit ANK-strepII, zeugt von der Bedeutung der Schritt 3.1 vor der Denaturierung (Spuren 7 und 8).

<ol>
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T., Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2011).</li><li>Tzfira, T., Vaidya, M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIP1,+an+Arabidopsis+protein+that+interacts+with+Agrobacterium+VirE2,+is+involved+in+VirE2+nuclear+import+and+Agrobacterium+infectivity.">VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.</a> EMBO J. 20, 3596-3607 (2001).</li><li>Chen, M. H., Sheng, J., Hind, G., Handa, A., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+the+tobacco+mosaic+virus+movement+protein+and+host+cell+pectin+methylesterases+is+required+for+viral+cell-to-cell+movement.">Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.</a> EMBO J. 19, 913-920 (2000).</li></ol>

Keine Interessenskonflikte erklärt.

Dieser Ansatz eignet sich zur Prüfung Protein-Protein-Wechselwirkungen zwischen Kombinationen der Proteine, wenn mindestens eine von denen die Proteine ​​ist leicht löslich in den Bindungspuffer und erfolgreich an andere Kombination von Proteinen 17,18 aufgebracht. Die iInteractions zwischen den Proteinen, die sowohl unter diesen Bedingungen unlöslich kann nicht durch dieses Protokoll getestet werden. Auch erfolgreiche Rückfaltung von ProIM kritisch für den Test. Spülen ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Protein-Assay-Kit </td><td> Bio-Rad </td><td> 500-0001 </td></tr><tr><td> Proteinase-Inhibitor-Cocktail </td><td> SIGMA </td><td> S8820 </td></tr><tr><td> Mini-PROTEAN System </td><td> Bio-RAD </td><td> 165-8000 </td></tr><tr><td> Semi-dry Western-Blot SD Elektrotransfer System </td><td> Bio-RAD </td><td> 170-3940 </td></tr><tr><td> Anti-Kaninchen-IgG-Antikörper, konjugiert mit Meerrettichperoxidase </td><td> GenScript </td><td> A00098 </td></tr><tr><td> Anti-GST polyklonalen Kaninchen-Antikörper </td><td> GenScript </td><td> A00097 </td></tr><tr><td> Anti-strepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> BioTrace, NT Nitrocellulose Transfer Membran </td><td> Pall Scientific </td><td> 27377-000 </td></tr><tr><td> Immobilon westlichen Chemilumineszenz HRP Substrat </td><td> Millipore </td><td> WBKL S0 050 </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

Validating Interaktionen zwischen verschiedenen Proteinen ist von entscheidender Bedeutung für die Untersuchung ihrer biologischen Funktionen auf molekularer Ebene. Es gibt mehrere Methoden, sowohl in vitro und in vivo, um Protein-Bindung zu bewerten, und mindestens zwei Methoden, die die Mängel der einander ergänzen sollten durchgeführt werden, um zuverlässige Erkenntnisse zu erhalten. Für eine in vivo-Assay, der bimolekularen Fluoreszenz Komplementation (BIFC)-Assay die beliebteste und am wenigsten invasive Ansatz, um Protein-Protein Interaktion in lebenden Zellen erkennen kann ist, zu identifizieren sowie die intrazelluläre Lokalisation der wechselwirkenden Proteinen 1,2. In diesem Test nicht-fluoreszierenden N-und C-terminalen Hälften von GFP oder seinen Varianten zu testen Proteine ​​fusioniert sind, und wenn die beiden Fusionsproteine ​​zusammen sind aufgrund der getesteten Proteine ​​Interaktionen gebracht wird, wird das Fluoreszenzsignal 3-6 rekonstituierte . Da das Signal ist leicht nachweisbar durch Epifluoreszenz oder konfokalen Mikroskopie hat BIFC als ein mächtiges Werkzeug der Wahl unter Zellbiologen zur Untersuchung über Protein-Protein-Interaktionen in lebenden Zellen 3 entstanden. Dieser Test kann jedoch manchmal zu falschen positiven Ergebnissen. Zum Beispiel kann das Fluoreszenzsignal durch zwei GFP-Fragmenten so weit wie 7 nm von einander durch so angeordnet, dass eine dichte Packung in einem kleinen subzellulären Kompartiment aufgelöst werden, sondern dass aufgrund der spezifischen Wechselwirkungen 7. Aufgrund dieser Einschränkungen sollten die Ergebnisse von Live Cell Imaging-Technologien erhalten von einem unabhängigen Ansatz, der auf einem anderen Prinzip zur Detektion von Protein-Interaktionen bestätigt werden. Co-Immunpräzipitation (Co-IP) oder Glutathion-Transferase (GST) Pull-down-Assays stellen solche alternative Methoden, die häufig verwendet werden, um Protein-Protein-Interaktionen in vitro zu analysieren. Allerdings bBei diesen Tests müssen jedoch die getesteten Proteine ​​werden leicht löslich in den Puffer, der für die Bindungsreaktion supportsused. Daher können keine speziellen Wechselwirkungen zwischen einem unlöslichen Protein nicht durch diese Techniken bewertet werden. Hier zeigen wir Ihnen das Protokoll für die Protein-Membran-Overlay-Bindungs-Assay, die diese Schwierigkeit umgeht. Bei dieser Technik kann die Interaktion zwischen löslichen und unlöslichen Proteinen zuverlässig geprüft werden, weil eines der Proteine ​​auf eine Membran-Matrix immobilisiert ist. Diese Methode, die in Kombination mit in vivo-Experimente, wie BIFC, bietet eine zuverlässige Methode zu untersuchen und zu charakterisieren Interaktionen treu zwischen löslichen und unlöslichen Proteinen. In diesem Artikel Bindung zwischen Tabak-Mosaik-Virus (TMV) Bewegung Protein (MP), die mehrere Funktionen ausübt während virale Zelle zu Zelle transportieren 8-14, und eine vor kurzem identifizierte Pflanze zellulären interactor, Tabak Ankyrin repeat-containing protein (ANK ) 15, wird gezeigt, mit dieser Technik.

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testen Protein-Protein-Interaktion ist für die Präparation von Protein-Funktionalität unverzichtbar. Hier stellen wir eine<em&gt; In-vitro-</em&gt; Protein-Protein-Bindungs-Assay auf eine Membran-immobilisierten Protein mit ein lösliches Protein-Sonde. Dieser Test liefert eine zuverlässige Methode, um die Interaktion zwischen einem unlöslichen Protein und ein Protein in Lösung zu testen.

Proteinmembran Overlay Assay: Ein Protokoll, die Interaktion zwischen löslichen und unlöslichen Proteinen testen In-vitro-</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

22.0K Aufrufe.  State University of New York. Testen Protein-Protein-Interaktion ist für die Präparation von Protein-Funktionalität unverzichtbar. Hier stellen wir eine In-vitro- Protein-Protein-Bindungs-Assay auf eine Membran-immobilisierten Protein mit ein lösliches Protein-Sonde. Dieser Test liefert eine zuverlässige Methode, um die Interaktion zwischen einem unlöslichen Protein und ein Protein in Lösung zu testen.

Serie: Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

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.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

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

Forschung

Biologie

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)

Bimolekularen Fluoreszenz Komplementation (BIFC) Assay für Protein-Protein-Wechselwirkungen in Onion Zellen unter Verwendung des 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 ...

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.

Eine flüssige Phase Affinity Capture Assay Verwendung von magnetischen Kügelchen, um Protein-Protein Interaktion Studie: Das Poliovirus-Nanobody Beispiel

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

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Einsatz eines Roboters für High-Throughput-Kristallisation von Membranproteinen in Lipidische Mesophasen

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In-vitro-Test zur Methylierung Protein-Arginin-Methylierung Studieren

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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).

Genomweite Protein-Protein-Wechselwirkungen durch Screening Protein-Fragment Komplementierungsassay (PKA) in lebenden Zellen

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

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.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

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

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase Komplementierung Imaging Assay in Nicotiana Benthamiana Blätter für Transient Bestimmung Proteinprotein Interaktion Dynamik

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

Bestimmung der dreigliedrigen Wechselwirkung zwischen zwei Monomeren eines MADS-Box-Transkriptionsfaktors und eines Calcium-Sensorproteins mittels BiFC-FRET-FLIM-Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

Herstellung und Bildgebung von rekombinanten proteingefüllten Vesikeln aus E. coli

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

Autor im Rampenlicht: Die Produktion rekombinanter Proteine 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.