1. Preparazione del campione di proteine <ol><li> In un tubo di 500 microlitri microcentrifuga, combinare i seguenti: </li></ol><table border="1"><tbody><tr><td> Reagente </td><td> Volume di aggiungere: </td></tr><tr><td> * Campione di proteine ​​(fino a 1 mg) </td><td> Fino a 112 microlitri </td></tr><tr><td> Esempio di Buffer (5X), fornito in kit di cartucce </td><td> 30 microlitri </td></tr><tr><td> DTT 1M </td><td> 8μL </td></tr><tr><td> 18 Ω H 2 O </td><td> x microlitri per un totale di volume del campione di 150μL ** </td></tr></tbody></table><ol start="2"><li> Denaturare i campioni a 95 ° C per 5 minuti. Raffreddare a temperatura ambiente. </li></ol> * L&#39;importo totale di proteine ​​da separare dipenderà dalla complessità del campione. Per i campioni più complessi, come il tessuto omogenato, 150-200 mcg per canale è raccomandato. ** Se il volume del campione è inferiore a 112 microlitri, aggiungere 18 Ω H 2 O per portare il volume finale a 150 microlitri. 2. Caricamento della cartuccia GELFREE <ol><li> Rimuovere il GELFREE 8100 cartuccia dalla confezione sigillata. </li><li> Rimuovere e gettare la pellicola sigillante. </li><li> Rimuovere il buffer di memoria dal scompartimenti cartuccia con una pipetta. Se tutti gli 8 camere di cartuccia sarà utilizzato, la cartuccia può essere invertita per esaurire il buffer di memorizzazione dal comparto. </li><li> Preparare la cartuccia per il caricamento con l&#39;aggiunta di GELFREE tampone di corsa a ciascuna delle camere di cartuccia come segue: </li></ol><table border="1"><tbody><tr><td> GELFREE camera di cartuccia </td><td> Volume di tampone GELFREE corsa per aggiungere </td></tr><tr><td> Serbatoio tampone anodo </td><td> 8 mL </td></tr><tr><td> Catodo serbatoio tampone </td><td> 6 ml </td></tr><tr><td> Raccolta camere </td><td> 100 l ciascuno </td></tr></tbody></table><ol start="5"><li> Rimuovere e scartare qualsiasi tampone dalla camera di caricamento dei campioni. </li><li> Caricare i 150 campioni microlitri nelle camere di carico con un 8-canali pipetta. </li><li> Posizionare la cartuccia caricata nella stazione 8100 Frazionamento GELFREE. Abbassare il schiere di elettrodi e chiudere il coperchio. </li></ol> 3. In esecuzione della cartuccia <ol><li> Utilizzando lo strumento display touch s schermo, premere il pulsante metodo per visualizzare l&#39;elenco dei metodi pre-programmati. Ciascuno dei metodi pre-programmati è composto da tensioni set e basata sul tempo pause progettato per risolvere le proteine ​​in un determinato intervallo di pesi molecolari. Il 8100 GELFREE automaticamente una pausa di raccolta delle frazioni agli intervalli specificati nel metodo. </li><li> Una volta che il metodo più appropriato è stato identificato, premere il pulsante di recuperare e inserire il numero desiderato di pre-programmati con il metodo di tastiera visualizzata sullo schermo. Premere ok, quindi premere il pulsante Applica. Il metodo è stato applicato verrà visualizzato nella parte inferiore della schermata principale. </li><li> Per selezionare i canali (numero di campioni) che verrà utilizzato nel lungo, premere il pulsante del canale nella schermata principale. Per selezionare tutti gli otto canali, premere selezionare tutte. Poi, premere fatto per tornare alla schermata principale. </li><li> Assicurarsi che il coperchio di protezione è chiuso e che la spia è verde nella parte inferiore dello schermo. </li><li> Per iniziare la separazione delle proteine, premere start. La Stazione Frazionamento automaticamente in pausa quando è il momento di raccogliere frazioni. Nota: Mentre il sistema è in esecuzione, i cerchi indicato nella casella sul lato sinistro dello schermo, raffigurano lo stato di ogni canale. Un cerchio verde indica che il canale è attivo, un cerchio giallo indica che lo strumento è in pausa, e un cerchio rosso indica che il canale non è riuscito. Le tensioni applicate e le correnti vengono visualizzati a destra dei cerchi lo status del canale. Le frazioni devono essere rimosso solo quando i cerchi di stato hanno trasformato un avviso giallo e il testo appare sullo schermo dello strumento s. </li><li> Per rimuovere le frazioni, aprire il coperchio dello strumento e utilizzare una pipetta a 8 canali di ritirare 150 microlitri di ciascuna delle camere di raccolta. </li><li> Una volta che le frazioni sono state rimosse, lavare la camera di raccolta GELFREE due volte con tampone di corsa con l&#39;aggiunta di 100 l per canale e pipettando su e giù due volte. </li><li> Dopo il lavaggio, aggiungere 100uL di GELFREE corsa tampone di nuovo nelle camere di raccolta, chiudere il coperchio e premere il pulsante Riprendi. </li><li> Il GELFREE 8100 durerà fino l&#39;intervallo di tempo successivo. Il tempo totale residuo nell&#39;esperimento viene visualizzata sul lato destro dello schermo. Per passare il tempo rimanente, il tempo trascorso e il tempo per mettere in pausa, premere il pulsante sopra la finestra di visualizzazione del tempo. </li><li> Una volta che tutte le frazioni sono state collected, possono essere utilizzati immediatamente per l&#39;ulteriore preparazione a valle, come isoelettrofocalizzazione o elettroforesi su gel, o per l&#39;analisi mediante spettrometria di massa, cromatografia liquida o immunoaffinità. </li></ol> 4. Rappresentante Risultati A 200 mg di omogenato di fegato bovino è stato suddiviso in 12 frazioni ciascuno, utilizzando il sistema GELFREE frazionamento. 6 ml di ogni frazione è stato eseguito su un normale gel 1D SDS PAGE e tinto d&#39;argento. Come mostrato nella Figura 1, il campione è stato frazionato in 12 frazioni distinte, che coprono la gamma di peso molecolare da 3.5kDa a 150kDa. Uno standard di peso molecolare (ColorBurst, Sigma) sono inoltre riportati in corsie 1 e 14. Il gel qui evidenzia l&#39;alto tasso di recupero realizzato utilizzando il sistema GELFREE 8100 per il frazionamento delle proteine. Il gel qui rappresenta il 4% delle proteine ​​recuperato da 200 mg. <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Le proteine ​​dal fegato bovino omogenato separati usando il sistema GELFREE Frazionamento. 6 ml di ogni frazione di 150 microlitri è stato caricato ed eseguito su un gel 1D SDS PAGE e tinto d&#39;argento. Un peso molecolare (MW) di serie è stato anche caricato come indicato. Si prega di <a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1.

<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>

Gli autori di questo articolo sono impiegati da Scoperta proteina che produce reagenti e strumenti utilizzati in questo articolo.

Il sistema 8100 Frazionamento GELFREE fornisce un rapido metodo semplice e riproducibile per il partizionamento campioni di proteine ​​complesse in discreto peso molecolare a base di frazioni. Questo nuovo metodo di frazionamento elimina la necessità di banda o il taglio di macchia, rendendo possibile isolare le proteine ​​in un ampio intervallo di massa in 90 minuti. La capacità di carico di quantità relativamente elevate di campioni e di recuperare frazioni in fase liquida è particolarmente utile in isolamento e l&#39;analisi delle proteine ​​a bassa abbondanza, che sono in genere più difficili da individuare. Le frazioni risultanti possono essere utilizzati in una vasta gamma di applicazioni, come LC-MS, MALDI-TOF, e immunoblotting.

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

Il sistema di frazionamento GELFREE 8100 è un nuovo sistema di frazionamento delle proteine ​​progettate per massimizzare il recupero delle proteine ​​durante il frazionamento peso molecolare basato. Il sistema è composto da monouso, 8-campione cartucce di capacità e un frazionamento da banco GELFREE strumento. Durante la separazione, una tensione costante viene applicata tra anodo e catodo serbatoi, e ogni miscela proteica è elettroforeticamente guidato da un vano di carico in un gel gel appositamente progettato colonna. Le proteine ​​sono concentrati in una fascia stretta in un gel accatastamento, e separati in base al loro rispettivo mobilità elettroforetica in un gel risolvere. Le proteine ​​eluire dalla colonna, sono intrappolati e concentrato in fase liquida nella camera di raccolta, libero del gel. Lo strumento viene poi messo in pausa a intervalli di tempo specifici, e le frazioni vengono raccolte con una pipetta. Questo processo viene ripetuto fino a quando tutte le frazioni desiderati sono stati raccolti. Se meno di 8 campioni sono eseguiti su una cartuccia, le camere a inutilizzati possono essere utilizzati in separazioni successive. Questa nuova tecnologia consente la separazione semplice e veloce fino a 8 miscele di proteine ​​complesse contemporaneamente, e offre diversi vantaggi rispetto ai metodi di frazionamento precedentemente disponibili. Questo sistema è in grado di frazionamento fino a 1 mg di proteine ​​totali per canale, per un totale di 8 mg per cartuccia. Proteine ​​intatte su una vasta gamma di massa sono separate sulla base del peso molecolare, mantenendo importanti proprietà fisico-chimiche dell&#39;analita. L&#39;intrappolamento fase liquida prevede il recupero elevati, mentre eliminando la necessità di banda o taglio spot, rendendo il processo di frazionamento altamente riproducibili 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

Il video che accompagna descrive l&#39;uso del Sistema di 8100 Frazionamento GELFREE, che partizioni campioni di proteine ​​complesse, sulla base del peso molecolare e recupera le frazioni in fase liquida. Il video descrive come funziona la tecnologia, come vengono utilizzati, e fornisce dati che ne risulta, con l&#39;analisi di elettroforesi su gel di poliacrilamide frazionato omogenato di fegato bovino.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

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

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JoVE Journal

Biology

16.3K Views.  Protein Discovery, Inc.. Il video che accompagna descrive l'uso del Sistema di 8100 Frazionamento GELFREE, che partizioni campioni di proteine ​​complesse, sulla base del peso molecolare e recupera le frazioni in fase liquida. Il video descrive come funziona la tecnologia, come vengono utilizzati, e fornisce dati che ne risulta, con l'analisi di elettroforesi su gel di poliacrilamide frazionato omogenato di fegato bovino.

Video: 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.

Preparazione e frazionamento di Xenopus laevis Egg Estratti

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

Ricerca

Biologia

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 Frazionamento subcellulare di cellule di mammifero aderenti e non aderenti

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.

Visualizzazione di Caenorhabditis Elegans Strutture cuticola Utilizzando il Dye lipofili Vital, 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 dispositivo per il recupero di campioni crioconservati cadere accidentalmente nelle cisterne criogeniche

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.

Un liquido fase di cattura test affinità utilizzando biglie magnetiche per studiare interazioni proteina-proteina: il poliovirus-Nanobody Esempio

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.

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del<em&gt; Arabidopsis thaliana</em&gt; Culture sospensione cellulare

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.

Polisoma Frazionamento e analisi dei mammiferi Translatomes su scala Genome-wide

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.

Studiando la proteina del sistema di controllo di qualità di<em&gt; D. discoideum</em&gt; Uso Cellulare Imaging a temperatura controllata

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.

Frazionamento subcellulare per l'attivazione di ERK previo trattamento di Peptide derivato mitocondriale

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.

Involucro di grafene di cellule di mammiferi fissate chimicamente per la microscopia elettronica a fase liquida

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