1. Beispiel Protein Preparation <ol><li> In einem 500 ul Mikrozentrifugenröhrchen, kombinieren Sie die folgenden: </li></ol><table border="1"><tbody><tr><td> Reagens </td><td> Volume hinzuzufügen: </td></tr><tr><td> * Protein-Probe (bis zu 1 mg) </td><td> Bis zu 112 ul </td></tr><tr><td> Sample Buffer (5fach) in Einbausatz zur Verfügung gestellt </td><td> 30 ul </td></tr><tr><td> DTT 1M </td><td> 8μL </td></tr><tr><td> 18 Ω H 2 O </td><td> x ul auf insgesamt Probenvolumen von 150 &amp;mgr; l ** </td></tr></tbody></table><ol start="2"><li> Denaturieren die Proben bei 95 ° C für 5 Minuten. Auf Raumtemperatur abkühlen. </li></ol> * Die Gesamtmenge an Protein zu trennen sein wird von der Komplexität der Probe ab. Für komplexere Proben, wie Gewebehomogenat, ist 150-200 g pro Kanal empfohlen. ** Wenn das Probenvolumen weniger als 112 ul ist, hinzufügen seit 18 Ω H 2 O zu einem Endvolumen von 150 ul zu bringen. 2. Lädt die GELFREE Cartridge <ol><li> Entfernen Sie die GELFREE 8100 Cartridge aus dem Folienbeutel. </li><li> Entfernen und Entsorgen der Abklebefolie. </li><li> Entfernen Sie die Speicher-Puffer aus der Kartusche Fächer mit einer Pipette. Wenn alle 8 Kammern der Kartusche verwendet wird, kann die Patrone invertiert, um die Speicher-Puffer aus den Fächern abzulassen. </li><li> Bereiten Sie die Patrone für das Laden, indem GELFREE Laufpuffer zu jedem der Patrone Kammern wie folgt: </li></ol><table border="1"><tbody><tr><td> GELFREE Patroneraumes </td><td> Volumen der GELFREE Laufpuffer hinzufügen </td></tr><tr><td> Anode Pufferspeicher </td><td> 8 mL </td></tr><tr><td> Cathode Pufferspeicher </td><td> 6 mL </td></tr><tr><td> Sammelkammern </td><td> 100 ul jeder </td></tr></tbody></table><ol start="5"><li> Entfernen Sie alle Puffer aus der Probe Füllraum. </li><li> Laden Sie die 150 ul Proben in die Be-Kammern mit einem 8-Kanal-Pipette. </li><li> Legen Sie das geladene Patrone in den GELFREE 8100 Fraktionierung Station. Senken Sie die Elektroden-Arrays und den Deckel schließen. </li></ol> 3. Laufen Sie die Patrone <ol><li> Mit dem Instrument s-Touchscreen-Display, drücken Sie die Methode, um die Liste der vorprogrammierten Methoden zu sehen. Jeder der vorprogrammierten Methoden besteht aus festgelegten Spannungen und zeitbasierte Pausen entwickelt, um Proteine ​​in einem bestimmten Bereich von Molekulargewichten zu lösen. Die GELFREE 8100 wird automatisch Pause für Fraktionssammlung in den Abständen in der angegebenen Methode. </li><li> Sobald die geeignete Methode identifiziert worden ist, drücken Sie die Taste abrufen, und geben Sie die Nummer des gewünschten vorprogrammierten Verfahren unter Verwendung der On-Screen-Tastatur. Drücken Sie OK, drücken Sie die Schaltfläche Übernehmen. Die Methode, die angewendet hat, wird am unteren Rand des Haupt-Bildschirm. </li><li> So wählen Sie die Kanäle (Anzahl der Proben), die im Vorfeld eingesetzt werden, drücken Sie die Kanal-Taste auf dem Hauptbildschirm. So wählen Sie alle acht Kanäle wählen, drücken Sie alle. Drücken Sie dann getan werden, um zum Hauptbildschirm zurückzukehren. </li><li> Stellen Sie sicher, dass die Sicherheit Deckel geschlossen ist und dass die LED-Anzeige befindet sich am unteren Rand des Bildschirms grün. </li><li> Zu Beginn der Protein-Trennung, den Startknopf drücken. Die Fraktionierung Station wird automatisch unterbrochen, wenn es Zeit Fraktionen Hinweis sammeln:. Während das System läuft, zeigen die Kreise in der Box auf der linken Seite des Bildschirms angezeigt den Status der einzelnen Kanäle. Ein grüner Kreis bedeutet, dass der Kanal aktiv ist, zeigt ein gelber Kreis, dass das Gerät angehalten wurde, und ein roter Kreis zeigt an, dass der Kanal ist ausgefallen. Die angelegten Spannungen und Ströme sind auf der rechten Seite des Kanals Status Kreise dargestellt. Fraktionen sollten nur entfernt, wenn der Status Kreise gedreht haben gelbe und eine Text-Alarm erscheint auf dem Instrument s Bildschirm. </li><li> Zum Entfernen Fraktionen, öffnen Sie den Deckel des Gerätes und die Verwendung einer 8-Kanal Pipette zu 150 ul aus jeder der Sammelkammern zurückzutreten. </li><li> Nachdem die Fraktionen entfernt worden sind, waschen Sie die Sammelkammer zweimal mit GELFREE Laufpuffer durch Zugabe von 100 ul pro Kanal und Auf-und Abpipettieren zweimal. </li><li> Nach dem Waschen, fügen Sie 100 ul der GELFREE Laufpuffer zurück in die Sammlung Kammern, den Deckel schließen, und drücken Sie die Wiederaufnahme-Taste. </li><li> Die GELFREE 8100 wird bis zum nächsten Zeitintervall ausgeführt werden. Die gesamte verbleibende Zeit in das Experiment auf der rechten Seite des Bildschirms gezeigt. Um zwischen verbleibende Zeit wechseln, verstrichene Zeit, und Zeit zu pausieren, drücken Sie die Taste über der Box Anzeigedauer einstellen. </li><li> Sobald alle Fraktionen wurden collected, können sie sofort zur Weiterverarbeitung Vorbereitung, wie isoelektrische Fokussierung oder Gel-Elektrophorese, oder für die Analyse mittels Flüssigchromatographie-Massenspektrometrie oder Immunaffinitäts verwendet werden. </li></ol> 4. Repräsentative Ergebnisse A 200 ug Rinderleber Homogenat wurde in 12 Fraktionen zu je partitioniert, mit dem GELFREE Fraktionierung System. 6 &amp;mgr; l jeder Fraktion wurde auf einem Standard-1D-SDS-PAGE-Gel und Silber gefärbt laufen. Wie in Abbildung 1 gezeigt, wurde die Proteinprobe fraktioniert in 12 verschiedene Fraktionen, überspannt den Molekulargewichtsbereich von 3.5kDa zu 150kDa. Ein Molekulargewicht-Standard (ColorBurst, Sigma) werden auch in den Spuren 1 und 14 dargestellt. Das Gel hier gezeigten Highlights der High-Recovery Rate erreicht mit dem GELFREE 8100 für Protein-Fraktionierung. Das Gel hier gezeigten stellt 4% des Proteins aus 200 ug erholt. <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="Abbildung 1" border="0" /> Abbildung 1. Proteine ​​aus Rinderleber Homogenat getrennt mit dem GELFREE Fraktionierung System. 6 &amp;mgr; l je 150 ul Bruchteil geladen wurde und auf einem 1D-SDS-PAGE-Gel und Silber gefärbt. Ein Molekulargewicht (MW)-Standard wurde ebenfalls geladen, wie angegeben. Bitte <a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 1 zu sehen.

<ol>
	<li>Tran, J. C., Doucette, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Size+Separation+of+Intact+Proteins+in+Solution+Phase+for+Mass+Spectrometry.">Multiplexed Size Separation of Intact Proteins in Solution Phase for Mass Spectrometry.</a> Anal Chem.. , (2009).</li></ol>

The authors of this article are employed by Protein Discovery that produces reagents and instruments used in this article.

Die GELFREE 8100 Fraktionierung System bietet eine schnelle, einfache und reproduzierbare Methode zur Partitionierung komplexer Protein-Proben in diskrete Molekulargewicht-basierte Fraktionen. Diese neuartige Methode der Fraktionierung entfällt die Notwendigkeit für Band-oder Spot-Schneiden, die es ermöglichen, Proteine ​​über einen weiten Massenbereich isolieren in 90 Minuten. Die Möglichkeit, relativ große Mengen an Proben laden und zu erholen Fraktionen in der flüssigen Phase ist besonders nützlich bei der Isolierung und Analyse von low-Fülle Proteine, die in der Regel schwieriger zu erkennen. Die erhaltenen Fraktionen können dann in eine breite Palette von Anwendungen, wie zB LC-MS, MALDI-TOF, und Immunoblot verwendet werden.

using the gelfree 8100 fractionation system for molecular weight-based fractionation with liquid phase recovery

Die GELFREE 8100 Fraktionierung System ist ein neuartiges Protein-Fraktionierung System entwickelt, um Protein-Rückgewinnung beim Molekulargewicht basierte Fraktionierung zu maximieren. Das System ist von Einweg-, 8-Probe-Tintenpatronen und eine Tisch-GELFREE Fraktionierung Instrument zusammen. Während der Trennung wird eine konstante Spannung zwischen Anode und Kathode Stauseen angelegt, und jeder Protein-Mischung wird elektrophoretisch aus einem Füllraum in eine speziell entwickelte Gel-Säule Gel getrieben. Proteine ​​sind in einem engen Band in einem Sammelgel konzentriert und getrennt auf der Grundlage ihrer jeweiligen elektrophoretischen Mobilitäten in einem Trenngel. Da Proteine ​​aus der Säule zu eluieren, werden sie gefangen und konzentriert in der flüssigen Phase in den Sammelraum, frei von dem Gel. Das Gerät wird dann in bestimmten Zeitabständen unterbrochen, und die Fraktionen werden gesammelt mit einer Pipette. Dieser Vorgang wird solange wiederholt, bis alle gewünschten Fraktionen gesammelt wurden. Wenn weniger als 8 Proben auf einer Kassette ausgeführt werden, können Sie unbenutzte Kammern in späteren Trennungen verwendet werden. Diese neuartige Technologie ermöglicht die schnelle und einfache Trennung von bis zu 8 komplexen Proteingemischen gleichzeitig und bietet mehrere Vorteile gegenüber bisher verfügbaren Fraktionierung Methoden verglichen. Dieses System ist in der Lage Fraktionierung bis zu 1 mg Gesamtprotein pro Kanal, also insgesamt 8 mg pro Patrone. Intakte Proteine ​​über einen weiten Massenbereich werden auf der Grundlage von Molekulargewicht aufgetrennt, Beibehaltung wichtiger physikalisch-chemischen Eigenschaften des Analyten. Die flüssige Phase Einschluss sorgt für hohe Recovery und eliminiert die Notwendigkeit für Band-oder Spot-Schneiden, so dass die Zerlegung in hohem Maße reproduzierbar 1.

Watch this Scientific Journal Video about Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery at JoVE.com

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

Das dazugehörige Video wird die Verwendung der GELFREE 8100 Fraktionierung System, welche Partitionen komplexen Protein-Proben auf der Basis von Molekulargewicht und erholt sich die Fraktionen in der flüssigen Phase. Das Video beschreibt, wie die Technologie funktioniert und wie es verwendet wird, und stellt daraus resultierenden Daten mit Polyacrylamidgelelektrophorese Analyse der fraktionierten Rinder Leberhomogenat.

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

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

using-gelfree-8100-fractionation-system-for-molecular-weight-based

Research

JoVE Journal

Biology

16.3K Aufrufe.  Protein Discovery, Inc.. Das dazugehörige Video wird die Verwendung der GELFREE 8100 Fraktionierung System, welche Partitionen komplexen Protein-Proben auf der Basis von Molekulargewicht und erholt sich die Fraktionen in der flüssigen Phase. Das Video beschreibt, wie die Technologie funktioniert und wie es verwendet wird, und stellt daraus resultierenden Daten mit Polyacrylamidgelelektrophorese Analyse der fraktionierten Rinder Leberhomogenat.

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

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Vorbereitung und Fraktionierung von Xenopus laevis Egg Auszüge

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Forschung

Biologie

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

In situ subzelluläre Fraktionierung von adhärenten und nicht adhärenten Säugetierzellen

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Visualisierung von Caenorhabditis elegans Cuticle Strukturen mit der lipophilen Vital Dye, DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Einrichtung zur Rückgewinnung von kryokonservierten Proben versehentlich fallengelassen in Kryo-Lagerbehälter

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabolische Markierung und Membran Fraktionierung für Vergleichende Proteomanalyse von<em&gt; Arabidopsis thaliana</em&gt; Fahrwerk Zellkulturen

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Polysomprofile Fraktionierung und Analyse von Säuger Translatomes auf einer Genom-weite Skala

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Die Untersuchung der Proteinqualitätskontrolle System der<em&gt; D. discoideum</em&gt; Verwendung von Temperaturgesteuerte Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the mitochondrial genome. MDPs have a wide variety of biological effects such as protecting neurons from apoptosis, improving metabolic markers, and protecting cells from chemotherapy. Humanin was the first MDP to be discovered and is the most studied peptide among the MDP family. The membrane receptors and downstream signaling pathways of humanin have been carefully characterized. Additional MDPs such as MOTS-c and SHLP1-6 have been more recently discovered and the signaling mechanisms have yet to be elucidated. Here we describe a cell culture based method to determine the function of these peptides. In particular, cell fractionation techniques in combination with western blotting allow for the quantitative determination of activation and translocation of important signaling molecule. While there are other methods of cell fractionation, the one described here is an easy and straightforward method. These methods can be used to further elucidate the mechanism of action of these peptides and other therapeutic agents.

This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins.

Subzellulären Fraktionierung für ERK-Aktivierung bei der mitochondrialen abgeleitet Peptid-Behandlung

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

Graphengehäuse chemisch fixierter Säugetierzellen für die Flüssigphasenelektronenmikroskopie

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...