Name: a method for extracting pigments from squid doryteuthis pealeii
Uploaded: 2016-11-09T15:00:00.000+00:00
Duration: 11 min 3 s
Description: Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

Invertebrate Tierstudien durchgeführt, hier sind in den Vereinigten Staaten nicht geregelt; daher die Institutional Animal Care und Use Committee hat keine Befugnis zur Überprüfung solcher Protokolle. Anstelle dieser nicht unter die Zuständigkeit der Regulierung in den Vereinigten Staaten fallen, so die Autoren hiermit, dass diese Studien wurden mit aufrichtigen Bemühungen im Hinblick auf die ethische Nutzung, Pflege und Behandlung dieser Tiere durchgeführt wird, wurde die Anzahl der Individuen minimiert und diese Bemühungen mit der Basler Deklaration und des Internationalen Rates für Versuchstierkunde (ICLAS) Ethik-Richtlinien stehen im Einklang. 1. Doryteuthis pealeii (D. pealeii) Dissection <ol><li> Kauf enthauptet Tintenfisch D. pealeii von der Marine Ressourcen Abteilung am Marine Biological Laboratory in Woods Hole, MA oder einer Frischfischmarkt. Verwenden Sie keine Proben, die um verrundet oder zuvor geändert wurden, um dieses Verfahren anzuwenden. Insbesondere ist die Chromatophore Schicht muss noch auf die Probe intakt sein. <ol><li> Wenn die Tiere nicht sofort verwendet werden, lagern Sie sie bei -20 ° C bis zur Verwendung. Um die Proben aufzutauen, legen Sie sie in Wasser bei Raumtemperatur für 1 Stunde vor der Präparation. </li></ol></li><li> Mit Edelstahl gerade fein-Punkt-Dissektion Schere entlang der hinteren Seite des Tieres geschnitten. Beginnen an der Basis des ventralen Mantel und an dem Rippenbereich endet. </li><li> Entfernen Sie alle inneren Organe (Magen, Kiemen, Tinte Drüse, Fortpflanzungsorgane, systemische Herz und brachial Herz) von ihnen heben mit einer Pinzette zu sezieren und Abtrennen ihre Bindegewebe Dissektion Schere. Entsorgen Organe in einem Probenbeutel. </li><li> Verwenden Sie sezieren T-Pins den Mantel an Pin (fin Seite nach oben) in einem Standard-11-1 / 2 &quot;x 7-1 / 2&quot; x 1-1 / 2 &quot;pan Aluminium Dissektion eine flexible Präparation Pad enthält. Versenken des Gewebes in 250 ml Meerwasser, das durch eine Einweg-Polystyrol-Vakuum-Filtrationssystem gefiltert wurdeenthaltend 0,2 &amp; mgr; m Porengrößen. </li><li> den epidermalen Hautschicht (Deckfarbe) mit Pinzette, die Chromatophoren haltige Hautschicht intakt bleibt vorsichtig entfernen. Sobald die epidermale Schicht entfernt wird, entsorgen Sie sie in einem Probenbeutel. </li><li> Präparieren klein (1 cm x 1 cm) Abschnitte der Chromatophoren-Schicht mit einer Pinzette und Schere Dissektion. Die Proben werden in 1,5 ml Mikrozentrifugenröhrchen (füllen die Hälfte jedes Leerrohr mit dem sezierten Gewebe). </li><li> Hinzufügen 500 ul gefiltertes Seewasser mit dem Gewebe enthaltenden Röhrchen. Die Proben können über Nacht bei 4 ° C gelagert werden. </li></ol> 2. Isolierkanne Chromatophor Pigmentgranula <ol><li> Tissue Verdauung <ol><li> Herstellung von Papain / Kollagenase <ol><li> Man wiegt 30 mg Kollagenase und 5 mg Papain mit einem 4 &quot;x 4&quot; niedrigen Stickstoff wiegen Papier. Bringen Sie diese in einem 50 ml-Polypropylen-Zentrifugenröhrchen mit Schraubverschluss. </li><li> Messen Sie 10 ml deionized Wasser mit einer Messpipette. Fügen Sie direkt Papain / Kollagenase Fläschchen enthält. Vortex auf die höchste Einstellung, bis die Lösung klar ist. </li></ol></li><li> Entfernen extrazelluläre Anhäufung <ol><li> Zentrifugieren des gesammelten chromatophore Gewebe aus Schritt 1.7 für 5 min bei 14.000 x g. Entsorgen Sie die obere Schicht aus Meerwasser in einen Kunststoffabfallbehälter. </li><li> In 500 ul der Papain / Kollagenase-Lösung auf das Gewebe ein P1000 variablen Volumen Einkanal-Mikropipette. Vortex jede Probe für 1-2 Minuten auf die höchste Einstellung. </li><li> Beschallen die Proben 5 min bei einer Frequenz von 40 kHz Zellen aus dem umgebenden Gewebe zu trennen. </li><li> Zentrifuge für 5 min bei 14.000 x g. Entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter. </li><li> Zentrifugieren des gesammelten chromatophore Gewebe für 5 min bei 14.000 x g. Entsorgen Sie die obere Schicht aus Meerwasser in einen Kunststoffabfallbehälter. Wiederholen Sie noch einmal. </li><li> Entfernen Sie manuell alle verbleibenden großen Gewebe secgen, die in diesem Prozess mit einer Pinzette verdauen nicht vor dem nächsten Schritt fortfahren. </li></ol></li></ol></li><li> Isolieren der Pigmentkörnchen <ol><li> Herstellung von Homogenisierungspuffer <ol><li> Wiegen 2,38 g (4- (2-Hydroxyethyl) -1-piperazinethansulfonsäure) (HEPES, 100 mM), 0.095 g Magnesiumchlorid (10 mM), 0,856 g Kalium-aspartat (50 mM) und 0,015 g Dithiothreitol (1 mM). In einen 150 ml Polystyrolbehälter mit Schraubverschluss. In einer kommerziellen Mini-Tablet-Protease-Inhibitor. </li><li> Messung der 100 ml entionisiertes Wasser mit einem Meßzylinder verwendet wird. Fügen Sie direkt in den Behälter, der die Homogenisierung Salze enthält. Vortex, bis die Lösung klar ist. </li></ol></li><li> Homogenisieren von Pigmentgranulaten <ol><li> Zentrifugieren jede Probe aus Schritt 2.1 bei 14000 × g für 5 min. Entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter. Diese Probe sollte frei von extrazellulären Gewebe sein. </li><li> In 500 &amp;# 181; l Homogenisierungspuffer in jedes Röhrchen und Vortex für 1-2 min je gründlich die Proben mischen. Beschallen die Proben für 30 Minuten (~ 40 kHz) und folgen mit 5 min Zentrifugation bei 14.000 x g. </li><li> Entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter, und wiederholen Sie 2-3 mal den vorherigen Schritt die vollständige Verdauung der chromatophore sacculus und Protein Anbindehaltung zu gewährleisten. Die verbleibende Lösung sollte eine hellgelbe Überstand enthalten und eine rot gefärbte Pellet die isolierten Pigmentkörnchen enthalten. </li></ol></li></ol></li></ol> 3. Pigmentextraktion <ol><li> Herstellung von saurem Methanol (HCl-MeOH) <ol><li> Vorsichtig 25 ul konzentriert (36,5 bis 38% (w / w)), Salzsäure (HCl) zu 5 ml Methanol (MeOH). Lagern Sie das HCl-MeOH-Lösung in einem Schraubdeckel Glasprobenfläschchen. Vorsichtig schwenken, um die Lösung gleichmäßig mischen. HINWEIS: Je saurer die Lösung ist, desto mehr Pigment wird während der fi extrahiert werdenerste Extraktion. </li></ol></li><li> Extraktion von Pigment <ol><li> Zentrifugieren Sie die Pigmentgranulatsuspension (von 2.2.2) für 5 min bei 14.000 xg und entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter. </li><li> In 500 ul der HCl-MeOH-Lösung und Vortex jede Probe für ~ 3-4 min. Beschallen die Proben für 10 min (~ 40 kHz), dann zentrifugieren für 5 min bei 14.000 x g. </li><li> Mit Hilfe einer Transferpipette, sammeln Sie den Überstand in einer 1-Dram-Schraubdeckel-Glasfläschchen. Der Überstand sollte in der Farbe dunkelrot sein. </li><li> Wiederholen Sie den Extraktionsschritt 3.2.2 bis keine weitere Farbe extrahiert (~ 4-5 Wäschen). HINWEIS: Jede Extraktion wird ~ 1,5 ml Pigment ergeben. Das verbleibende farblose Pellet wird das Pigment extrahierte Granulat. Hinweis: beide farblos Granulat und extrahiert Pigmente können in Wasser bei 4 ° C bis zur weiteren Verwendung gelagert werden. </li></ol></li></ol> 4. Charakterisierung gesamten Extraktionsverfahren <ol><li> Scannen Electron Mikroskopie <ol><li> Bild das Granulat (nicht umgesetztes aus Schritt 2.2.1 und pigment extrahiert aus Schritt 3.2.3) unter Verwendung von Rasterelektronenmikroskopie (SEM), um die Reinheit des Extraktionsprozesses zu überprüfen. </li><li> Um Granulatproben für die Bildgebung, erste Zentrifuge beide nicht umgesetzten Granulat und Pigment extrahiert Granulat für 5 min bei 14.000 · g, und entsorgen Sie den Überstand in ein Kunststoff-Abfallbehälter herzustellen. Re-suspendieren diese in 1 ml Ethanol und Ablagerung 2-3 Tropfen der Granulatsuspension auf Aluminium SEM Stubs eine Transferpipette. </li><li> über Nacht, nachdem die Proben trocknen, Sputter-Mantel mit Gold-Platin-Beschichtung für 3 min eine 15 nm Beschichtung zu erreichen. </li><li> Bild unter Verwendung eines SEM mit einer Schottky-Emitter und einem Sekundärelektronendetektor. Verwenden Sie einen 5-7 kV Strahlspannung und einen Arbeitsabstand zwischen 8 und 9 mm Bild diese Materialien. </li></ol></li><li> UV-VIS (UV-vis) spektrophotometrische Messungen <ol><li> Überwachen Sie die Absorption proDateien für alle drei Bedingungen (nicht umgesetztes Granulat aus 2.2.2, extrahiert Pigment aus 3.2.3 und Pigment extrahiert Granulat aus 3.2.4) mit einem Doppelstrahl-Spektrophotometer 200-800 nm verwendet wird. </li><li> Sammeln Sie eine Leermessung VE-Wasser in einer 1-ml-Küvette für den nicht umgesetzten Granulat und das Pigment extrahiert Granulat verwendet wird. Sammeln Sie UV-Vis-Absorptionsspektren für beide Proben. </li><li> Sammeln Sie eine leere Messung der sauren Methanollösung in einer 1-ml-Küvette für das extrahierte Pigment verwendet wird. Sammeln Sie UV-Vis-Absorptionsspektrum für die Probe. </li><li> Bestimmen maximale Wellenlänge jeder Probe durch die Wellenlänge identifiziert, an dem die Absorptionsspitze ihre maximale relative Höhe erreicht. </li></ol></li></ol>

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We have nothing to disclose.

Wir haben ein Verfahren zum Extrahieren von Pigmenten squid chromatophore Granulat demonstriert. Durch speziell auf die Granulat-Targeting, unser Ziel ist es, ihre Rolle bei der Vermittlung der adaptive Färbung zu bestimmen. Diese Methode unterscheidet sich von früheren Berichten entworfen Kopffüßer Pigmente mit Bulk - Gewebeproben 14 oder gefriergetrocknet Haut 15 zu charakterisieren. Während dieses Protokolls auf Extrahieren chromatophore Pigmente wirksam ist, wi...

Kopffüßer wie Tintenfische, Tintenfische und Kraken haben die Fähigkeit, dynamisch ihr Aussehen verändern , für Tarnen und Signalisierung. 1-6 Diese Fähigkeit durch die selektive Flächenausdehnung von pigmentierten Organe teilweise als Chromatophoren bekannt unterstützt wird. 4,7-9 Chromatophoren Weich Aktoren , die ein Netzwerk von nanostrukturiertem Pigmentgranulaten in einem cytoelastic sacculus beschränkt enthalten , die radial von Muskelfasern verankert ist. 1,3 Da sie betätigt werden, Chromatophoren um 500% in präsentierten Oberfläche erweitern , um die Granulate im gesamten Organ verteilen. 3,7 10,11, Wenn diese Aktion über eine Anzahl von aufeinander abgestimmten Chromatophoren wird, wird die Gesamtfärbung des Tieres verändert. Während es bekannt ist, daß die Pigmentgranulate dieser Farbänderung beitragen, bleibt deren Zusammensetzung unbekannt. Wir beschreiben ein Verfahren zur Isolierung und Reinigung von chromatophore Pigmente, die für zukünftige Studien Zusammensetzungs angepasst werden kann. 3,12 Chromatophor haltiges Gewebe durch sorgfältige Exstirpation von der Kopffüßer geerntet wird. Eine Verdauung und Homogenisierungsverfahren wird dann verwendet, um das umgebende Gewebe zu dissoziieren und die chromatophore Zellen trennen. Die nanostrukturierten Granula werden dann isoliert und gereinigt aus den verbleibenden Chromatophoren wiederholt unter Verwendung von Beschallung und Zentrifugation. Nach der Reinigung werden Pigmente aus dem Granulat in einem Verfahren extrahiert , das aus der Gewinnung von sichtbaren Farbe aus Schmetterlingsflügeln mit saurem Methanol - Lösungen. 13 Rasterelektronenmikroskopie (SEM) und Spektrometrie werden verwendet , um zu bestätigen , dass die Chromatophoren - Pigmente erfolgreich extrahiert angepasst ist , dieser Prozess. Diese Methode beschreibt die Isolierung von chromatophore Granulat, das verwendet wird, um die molekularen Beiträge zu c zu erkundenoloration in Kopffüßer. 12 kleine Molekül - Extraktionen von ganzen Tieren kann oft ein langer und mühsamer Prozess sein. Das Ziel hier ist es, zukünftige Forscher einer effektiven und einfachen Protokoll für den Erwerb von Pigmenten aus den nanostrukturierten Granulat in Kopffüßer zu informieren.

<table><tbody><tr><td>Collagenase</td><td>Alfa Aesar</td><td>9001-12-1</td><td>No hazard</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3482-12-3</td><td>Irritant, acute toxicity</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td>Mild irritant</td></tr><tr><td>Hydrochloric acid</td><td>EMD Chemicals</td><td>7647-01-0</td><td>Corrosive</td></tr><tr><td>k-Aspartate</td><td>Sigma-Aldrich</td><td>1115-63-5</td><td>Reacts violently with oxidants</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Mild eye irritant</td></tr><tr><td>Methanol</td><td>Pharmco-AAPER</td><td>67-56-1</td><td>Highly flammable</td></tr><tr><td>Mini tablet prtoease inhibitor</td><td>Sigma-Aldrich</td><td>469315-90-01</td><td>Corrosive to metal and skin</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>9001-73-4</td><td>Irritant&amp;nbsp;</td></tr></tbody></table>

a method for extracting pigments from squid doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

Chromatophoren werden aus dem D. seziert pealeii dorsal Mantel (1A, 1B). Sobald sie entfernt werden, werden Chromatophoren lysiert und gereinigt Zyklen Zentrifugation und Waschen der pigmentierten Granulate (2A, 2B) zu isolieren geführt. Sauren Methanollösungen (HCl-MeOH) werden verwendet , um das Pigment aus dem Granulat (2C) zu extrahieren, um ein lösliches Pigment Extrakt und unl?...

Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Ein Protokoll für die Extraktion von Pigmenten aus den nanostrukturierten Granulat in Tintenfisch Doryteuthis pealeii Chromatophoren vorgestellt.

Ein Verfahren zum Extrahieren Pigmente aus Squid<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

a-method-for-extracting-pigments-from-squid-doryteuthis-pealeii

Research

JoVE Journal

Biology

A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K Aufrufe.  University of New Hampshire.  Ein Protokoll für die Extraktion von Pigmenten aus den nanostrukturierten Granulat in Tintenfisch Doryteuthis pealeii Chromatophoren vorgestellt. 

Serie: A Method for Extracting Pigments from Squid Doryteuthis pealeii

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Ein Fisch-Fütterung Labor Bioassay die antipredatory Aktivität von Sekundärmetaboliten aus dem Gewebe mariner Organismen beurteilen

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Forschung

Umwelt

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Quantifizierung der Bauchpigmentierung in<em&gt; Drosophila melanogaster</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

Genetik

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Wasser in Öl-Emulsionen: Ein neues System für die Montage wasserlösliche Chlorophyll-bindenden Proteine ​​mit hydrophober Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Chemie

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

Isolieren und die Einbeziehung von Licht-ernten Antennen von Diatomee Cyclotella Meneghiniana in Liposomen mit Thylakoidmembran Lipide

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

Biochemie

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Reinigung von Transcripts und Metaboliten aus<em&gt; Drosophila</em&gt; Köpfe

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Biologie

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In-vitro-Rekonstitution der Lichtsammelkomplexe der Pflanzen und Grünalgen

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

Methoden zur Inszenierung Pupal Perioden und Messung der Flügel Pigmentierung der Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Umgekehrter genetischer Ansatz zur Identifizierung von Regulatoren der Pigmentierung anhand von Zebrafischen

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

Ein Verfahren zum Extrahieren Pigmente aus Squid

Zusammenfassung

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Isolieren und die Einbeziehung von Licht-ernten Antennen von Diatomee Cyclotella Meneghiniana in Liposomen mit Thylakoidmembran Lipide

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

Reinigung von Transcripts und Metaboliten aus

In-vitro-Rekonstitution der Lichtsammelkomplexe der Pflanzen und Grünalgen

Methoden zur Inszenierung Pupal Perioden und Messung der Flügel Pigmentierung der Drosophila guttifera

Umgekehrter genetischer Ansatz zur Identifizierung von Regulatoren der Pigmentierung anhand von Zebrafischen

Ein Verfahren zum Extrahieren Pigmente aus Squid

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Ein Fisch-Fütterung Labor Bioassay die antipredatory Aktivität von Sekundärmetaboliten aus dem Gewebe mariner Organismen beurteilen

Quantifizierung der Bauchpigmentierung in

Wasser in Öl-Emulsionen: Ein neues System für die Montage wasserlösliche Chlorophyll-bindenden Proteine ​​mit hydrophober Pigments

Isolieren und die Einbeziehung von Licht-ernten Antennen von Diatomee Cyclotella Meneghiniana in Liposomen mit Thylakoidmembran Lipide

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

Reinigung von Transcripts und Metaboliten aus

In-vitro-Rekonstitution der Lichtsammelkomplexe der Pflanzen und Grünalgen

Methoden zur Inszenierung Pupal Perioden und Messung der Flügel Pigmentierung der Drosophila guttifera

Umgekehrter genetischer Ansatz zur Identifizierung von Regulatoren der Pigmentierung anhand von Zebrafischen

Wasser in Öl-Emulsionen: Ein neues System für die Montage wasserlösliche Chlorophyll-bindenden Proteine mit hydrophober Pigments