Questo lavoro è supportato da un finanziamento da US National Science Foundation a Christoph Benning.

1. Estrazione dei lipidi <ol><li> Iniziare l&#39;estrazione dei lipidi dalla raccolta di 30 mg di 4 settimane di età lascia Arabidopsis da piante coltivate su agar solidificato media o suolo e trasferirli in provette da 1,5 ml in polipropilene reazione. Foglie fresche possono essere istantaneamente congelati in azoto liquido e conservati a -80 ° C. </li><li> Aggiungere 300 microlitri di estrazione solvente composto da metanolo, cloroformio e acido formico (20:10:01, v / v / v) per ogni campione. Agitare vigorosamente (utilizzando una vernice shaker o simili) per 5 minuti. </li><li> Aggiungere 150 ml di acido fosforico 0,2 M (H 3 PO 4), 1 M di cloruro di potassio (KCl) e mescolare brevemente nel vortex. </li><li> Centrifugare a 13.000 xg a temperatura ambiente per 1 minuto. Lipidi sciolte nella fase cloroformio minore sarà individuato su piastre TLC. </li></ol> 2. Cromatografia su strato sottile (TLC) 15 <ol><li> Per preparare piatti TLC, immergere uno 20cmx20cm gel di silice rivestito con lastrina di caricamento striscia per 30 sec in 0,15 M di solfato di ammonio ((NH 4) 2 SO 4) soluzione, dopo immergendo per 30 secondi, asciugare la piastra per almeno 2 giorni un recipiente coperto. Durante l&#39;attivazione la sublimazione di ammonio lascia dietro di acido solforico, che protonates fosfatidilglicerolo necessarie per la sua separazione da glycerolipids altri. </li><li> Il giorno della sperimentazione, attivare TLC piastre di cottura in forno a 120 ° C per 2,5 ore. </li><li> Dopo il raffreddamento le piastre attivato a temperatura ambiente, usare una matita per disegnare una linea retta (1,5 cm dal bordo del piatto) attraverso la piastra all&#39;origine del cromatogramma. </li><li> In una cappa aspirante, lentamente consegnare 3 X 20 l di estratto lipidico in fase di cloroformio inferiore utilizzando una pipetta 20 microlitri con 200 ul punte di plastica gialla sotto una debole corrente di N 2. A tal fine, un tubo Tygon è collegata al regolatore del serbatoio 2 N. Mantenere il posto più piccolo di 1 cm di diametro. Ogni piatto può contenere fino a 10 campioni (quando successiva analisi GLC è prevista). </li><li> Come lipidi punti completamente asciutto nella cappa, preparare il solvente di sviluppo composto da acetone, toluene, acqua (91 ml: 30 ml: 7,5 ml). Se l&#39;umidità relativa dell&#39;aria è alta, la separazione potrebbe essere danneggiato. In questo caso l&#39;acqua deve essere ridotta a dare (91 ml: 30 ml: 7,0 mL) per ottenere la separazione desiderata. </li><li> Versare 80 ml solvente di sviluppo in un TLC sigillabile camera di sviluppo (L: H: W = 27,0: 26,5: 7,0 cm / cm / cm) e posizionare la lastra nella vasca con l&#39;estremità del campione verso il basso. Tenuta del serbatoio con il morsetto. Il solvente salirà il piatto e lipidi saranno separati. Il tempo di sviluppo è di circa 50 minuti a temperatura ambiente. </li><li> Quando il fronte del solvente ha raggiunto 1 cm dal bordo superiore della piastra, rimuovere con cautela la piastra dal serbatoio e asciugare completamente nella cappa per circa 10 minuti. </li><li> Lipidi separati da TLC può essere reversibile macchiato brevemente con iodio per l&#39;analisi quantitativa o irrimediabilmente macchiata con acido solforico o α-naftolo. <ol><li> Carbonizzazione acido solforico: spruzzare il piatto con il 50% di acido solforico in acqua in una bottiglia di vetro a spruzzo nella cappa e cuocere in forno a 120 ° C per 15 minuti (Figura 2A). </li><li> α-naftolo colorazione per glicolipidi: spruzzare il piatto con del 2,4% (w / v) α-naftolo nel 10% (v / v) di acido solforico, 80% (v / v) di etanolo e cuocere in forno a 120 ° C per 3-5 minuti fino a quando le bande glicolipidi sono macchiati rosa o viola (Figura 2B). Trattamento eccessivo porterà alla carbonizzazione di tutti i lipidi a causa della presenza di acido solforico nel reagente. </li><li> Colorazione di iodio: in una cappa aspirante, la piastra in una vasca TLC chiuso con cristalli di iodio (in un vassoio sul fondo portando alla saturazione dell&#39;atmosfera con vapori di iodio fino a quando i lipidi sono visibili). Non esporre la piastra di iodio troppo lungo lo iodio può modificare covalentemente acidi grassi polinsaturi (Figura 2C). In alternativa, per evitare l&#39;ossidazione dei lipidi, solo piste di serie intervallate da corsie campione deve essere colorato con una lana di vetro collegato pipetta Pasteur con cristalli di iodio attraverso la quale viene soffiata N 2 su singole corsie standard. </li></ol></li></ol> 3. Grassi Acil metilestere (FAME) di reazione 16 <ol><li> Rimuovere macchie di silice circostanti lipidi identificato dalla piastra TLC con una lametta. Raschiare la silice che contiene lipidi e trasferire la polvere di silice usando un imbuto in un tubo di vetro con un (PTFE)-tappo a vite rivestiti in Teflon. </li><li> Aggiungere 1 ml 1 N di acido cloridrico (HCl) in metanolo anidro di ogni campione con pipetta di vetro. </li><li> Aggiungere 100 ul 50 mg ml -1 pentadecanoic acido (15:00) con 200 microlitri pipetta ad ogni campione come standard interno con una pipetta 200 microlitri punta con 200 microlitri di plastica gialla. Mantenere un tubo con solo acido pentadecenoic in metanolica HCl come controllo. Chiudere ermeticamente con tubi di vetro rivestita di teflon tappi. </li><li> Incubare gltubi culo in un bagno d&#39;acqua 80 ° C per 25 minuti. Tubi devono essere sigillati in modo che il solvente non evapori. </li><li> Dopo che i tubi si sono raffreddati, aggiungere 1 ml di sodio cloruro 0,9% seguito da 1 ml di esano e vortice vigorosamente. Centrifugare i campioni a 1000xg per 3 minuti. </li><li> Nella cappa, rimuovere lo strato esano / superiore del campione con pipetta Pasteur e collocarlo in un nuovo tubo di vetro 13x100 millimetri. </li><li> Far evaporare esano sotto una debole corrente di N 2 senza seccare completamente. </li><li> Sciogliere il grasso risultante acil methylesters s in 60 microlitri esano. Trasferimento dei campioni in fiale autocampionatore e tappo. I campioni possono essere conservati a 4 ° C per breve termine e -20 ° C per pochi giorni. </li></ol> 4. Gascromatografia (GLC) 10 <ol><li> Prima di iniziare GLC, Assicurarsi che l&#39;elio, idrogeno e cilindri ad aria sono riempiti. </li><li> Esano sufficiente deve essere aggiunto al serbatoio del solvente ed il contenitore dei rifiuti deve essere vuoto. Per i grassi separazione acil methylesters, allegare un DB-23 colonna alla macchina. </li><li> Fiale posto nel campionatore automatico. Avviare il software Chemstation per GLC sul computer sistema. </li><li> Impostare la temperatura di ingresso a 250 ° C con una portata di elio a 48,6 ml min -1 e la pressione a 21,93 psi. Il rapporto di divisione è 30,0: 1. </li><li> La temperatura del forno è impostato inizialmente a 140 ° C per 2 minuti e cresciuto a 160 ° C ad una velocità di 25 ° C min -1. Quindi impostare la temperatura aumenterà da 160 ° C a 250 ° C ad una velocità di 8 ° C min -1 e tenere a 250 ° C per 4 minuti seguita da una diminuzione a 140 ° C ad una velocità di 38 ° C min - 1. Una corsa dura circa 21 minuti. </li><li> La temperatura del rivelatore a ionizzazione di fiamma è di 270 ° C con un tasso di idrogeno flusso di 30,0 ml min -1, portata d&#39;aria a 400 ml min -1 e la portata di elio al 30,0 ml min -1. </li><li> Inserire il numero di fiale e nomi di esempio nella tabella sequenza di esecuzione. Impostare il 10 L iniettore per iniettare un campione di 2 microlitri per flaconcino. </li><li> Quando lo strumento è pronto, iniziare la sequenza di esecuzione. </li></ol> 5. Rappresentante dei risultati: Esempi di colorazione irreversibile di TLC separati da lipidi da 4 settimane di età piantine di Arabidopsis sono mostrati nella Figura 2. I lipidi acido solforico colorato (Figura 2A) sono carbonizzati e appaiono come macchie scure. α-naftolo è preferito a macchia glicolipidi come MGDG, dgdg, SQDG glicolipidi ecc colorati con α-naftolo portare una rosa-viola, mentre gli altri lipidi polari macchia gialla (Figura 2B). La colorazione di iodio è reversibile e dà lipidi un colore giallastro che scompare in un tempo breve evapora iodio (Figura 2C). Lipidi iodio brevemente macchiato può essere sottoposto ad analisi GLC lipidi senza macchia, anche se sono preferibili per ridurre abbattere dei lipidi. Se fatto con successo, segnali distintivi che rappresentano differenti grassi acil metilico saranno osservati dopo GLC (Figura 3). Grassi metilico acilici con catena di carbonio più brevi e meno doppi legami hanno tempo di permanenza più brevi con la batteria DB-23 colonna. Grassi acil profiling metilico è uno strumento sensibile per identificare i mutanti con alterata composizione lipidica. Nella Figura 4, il MGDG18: 3 rapporto di acidi grassi molare è diminuito nel tgd4-1 mutante rispetto al wild-type 18. Dividendo il moli di grassi acil metilico per una classe di lipidi con le moli di tutte le classi di lipidi, il rapporto molare di ogni lipidi vengono calcolati. Ad esempio, per calcolare il rapporto molare di MGDG: (MGDG)% mol = Σ [FAME (MGDG)] / Σ [FAME (totale)] x100% I rapporti conseguenti molare di ogni classe dei lipidi sia dal tipo selvaggio e il mutante può essere paragonato. Per esempio, il tgd4-1 mutante è aumentata quantità relative di MGDG e PG, ma è diminuito quantità di dgdg e PE (Figura 5) 18. <img src="/files/ftp_upload/2518/2518fig1.jpg" alt="Figura 1" /> Figura 1. Diagramma di flusso di analisi dei lipidi polari utilizzando piantine di Arabidopsis. Lipidi totali sono estratti da 4 settimane di età piantine di Arabidopsis e separati da TLC. I lipidi separato può essere grattata da lastrina per transesterificazione seguita da analisi GLC. <img src="/files/ftp_upload/2518/2518fig2.jpg" alt="Figura 2" /> Figura 2. Separazione dei lipidi su lastre TLC. Estratti lipidici di 35 mg (peso vivo) piantine di tipo selvatico sono separati da TLC e macchiato da acido solforico (A), α-naftolo (B) o vapori di iodio (C). Tre ripetizioni sono indicati in ogni metodo di colorazione. Dgdg, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol, PC, fosfatidilcolina, PE, fosfatidiletanolamina, PG, phosphatidylglycerol, PI, fosfatidilinositolo, SQDG, sulfoquinovosyldiacylglycerol. <img src="/files/ftp_upload/2518/2518fig3.jpg" alt="Figura 3" /> Figura 3. Analisi GLC della Methylesters acidi grassi (FAME) derivato dal MGDG del tipo selvatico. FAMEs sono separati su una colonna di 30 m capillare e rilevati da ionizzazione di fiamma. Pentadecanoic acido (15:00) è utilizzato come standard interno. <img src="/files/ftp_upload/2518/2518fig4.jpg" alt="Figura 4" /> Figura 4. Profilo degli acidi grassi di MGDG nel tipo selvatico Col2 (colonne bianche) e la tgd4-1 mutante (colonne nere). Gli acidi grassi sono presentati come numero di atomi di carbonio, seguito dal numero di doppi legami. Tre ripetizioni viene calcolata la media e le deviazioni standard sono mostrati. <img src="/files/ftp_upload/2518/2518fig5.jpg" alt="Figura 5" /> Figura 5. Polare composizione dei lipidi del tipo selvatico Col2 (colonne bianche) e la tgd4-1 mutante (colonne nere). Tre ripetizioni viene calcolata la media e le deviazioni standard sono indicate dalle barre di errore.

<ol>
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Nessun conflitto di interessi dichiarati.

TLC accoppiato con GLC fornisce uno strumento robusto e rapido per l&#39;analisi quantitativa dei lipidi polari nelle piante. Piccoli cambiamenti nella composizione lipidica possono essere identificati, quindi, questo metodo è stato utilizzato per lo screening su larga scala di mutanti ridotta nei percorsi metabolici dei lipidi polari 1,20. Questo metodo è anche ampiamente utilizzato per attività di monitoraggio degli enzimi che utilizzano lipidi polari come substrato. 2,6,7 Oltre a foglie, la composizione lipidica di altri tessuti vegetali come radici e semi o frazioni subcellulari come i cloroplasti e mitocondri può essere determinata anche nello stesso modo. Il sistema di solventi (acetone, toluene, acqua) usato qui è ottimizzato per la separazione di glicolipidi e fosfolipidi nelle piante. Tuttavia, in tgd1, 2,3,4 mutanti e cloroplasti isolati, TGDG corre insieme al PE, mentre tetragalactosyldiacylglycerol funziona con PC. In questo caso un sistema di solvente con cloroformio, metanolo, acido acetico e acqua (85: 20: 10: 4, v / v / v / v) viene utilizzato 13. A volte bidimensionale TLC utilizzando due diversi sistemi solvente viene eseguita per separare ulteriormente glicolipidi e fosfolipidi 19. Inoltre, tessuti vegetali possono essere direttamente sottoposte alla reazione FAME seguito da GLC per determinare il profilo totale di acidi grassi, senza separazione iniziale sul TLC 5. Accanto alla TLC-GLC dimostrato sistema, un altro metodo utilizzato per la profilazione dei lipidi si basa sulla diretta di massa a ionizzazione electrospray tandem spettrometria di 17. In questo metodo la separazione cromatografica iniziale dei lipidi nel estratto è omesso. Tuttavia, questo metodo richiede apparecchiature costose e personale esperto, che lo rende meno utile per analisi di routine in laboratorio o per lo screening mutante.

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>&alpha;-naphthol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N1000</td>
<td>&nbsp;</td>
<tr>
<td>Methanolic HCL 3N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>33050-U</td>
<td>Dilute to 1N by methanol</td>
</tr>
<tr>
<td>Si250-PA TLC plates</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>7003-04</td>
<td>With pre-absorbent </td>
</tr>
<tr>
<td>TLC chamber</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Z266000</td>
<td>&nbsp;</td>
<tr>
<td>Screw cap tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53283-800</td>
<td>&nbsp;</td>
<tr>
<td>Scew caps</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>13-425</td>
<td>&nbsp;</td>
<tr>
<td>PTFE disk</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>200 608</td>
<td>&nbsp;</td>
<tr>
<td>GLC system</td>
<td>&nbsp;</td>
<td>Hewlett Packard</td>
<td>HP6890</td>
<td>&nbsp;</td>
<tr>
<td>DB-23 column</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>122-2332</td>
<td>&nbsp;</td>
<tr>
<td>GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>500 132</td>
<td>&nbsp;</td>
<tr>
<td>Caps of GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>201 828</td>
<td>&nbsp;</td>
<tr>
<td>Chemstation software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2070AA</td>
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arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (tlc) coupled with gas-liquid chromatography (glc)

Membrane biologiche delle cellule separato dall&#39;ambiente. Da una singola cellula per le piante e gli animali pluricellulari, glycerolipids, come fosfatidilcolina o fosfatidiletanolamina, membrane a doppio strato forma che fungono da entrambi i confini e interfacce per lo scambio chimico tra le cellule e l&#39;ambiente circostante. A differenza degli animali, cellule vegetali hanno un organello speciale per la fotosintesi, il cloroplasto. Il sistema a membrana intricato del cloroplasto contiene glycerolipids unico, vale a dire glicolipidi carente di fosforo: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (dgdg), sulfoquinovosyldiacylglycerol (SQDG) 4. I ruoli di questi lipidi sono oltre la semplice strutturali. Queste glicolipidi e glycerolipids altri sono stati trovati nelle strutture cristalline di fotosistema I e II che indicano il coinvolgimento di glycerolipids nella fotosintesi 8,11. Durante il digiuno fosfato, dgdg è trasferito alle membrane extraplastidic per compensare la perdita di fosfolipidi 9,12. Gran parte della nostra conoscenza della biosintesi e funzione di questi lipidi è stata tratta da una combinazione di studi genetici e biochimici con 14 Arabidopsis thaliana. Durante questi studi, una procedura semplice per l&#39;analisi dei lipidi polari è stato essenziale per lo screening e analisi di mutanti lipidi e saranno descritte in dettaglio. Un estratto lipidico foglia è prima separate da cromatografia su strato sottile (TLC) e glycerolipids sono macchiati reversibilmente con vapori di iodio. I lipidi individuali sono raschiati dalla piastra TLC e convertito in grasso methylesters acil (FAME), che vengono analizzati dal gas-cromatografia liquida accoppiata con rilevazione a ionizzazione di fiamma (FID-GLC) (Figura 1). Questo metodo ha dimostrato di essere uno strumento affidabile per lo screening dei mutanti. Ad esempio, il tgd1, 2,3,4 reticolo endoplasmatico-to-plastidiale mutanti traffico di lipidi sono stati scoperti in base all&#39;accumulo di un galactoglycerolipid anormale: trigalactosyldiacylglycerol (TGDG) e una diminuzione della quantità relativa di 18:3 (carboni: doppio obbligazioni) grassi gruppi acil in lipidi di membrana 3,13,18,20. Questo metodo è applicabile anche per determinare le attività enzimatiche di proteine ​​con lipidi come substrato 6.

Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composizione di estratti dei lipidi polari e la composizione in acidi grassi di glycerolipids individuali sono determinati in un esperimento lipidi semplice e robusto profilo. A tal fine, glycerolipids sono isolati per cromatografia su strato sottile e sottoposti a transmetilazione dei loro gruppi acilici. Grassi methylesters acil sono quantificati da gas-cromatografia in fase liquida.

Arabidopsis thaliana Profiling Glycerolipid Polar Cromatografia su strato sottile (TLC) Accoppiato con cromatografia gas-liquido (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

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Research

JoVE Journal

Biology

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

36.6K Views.  Michigan State University. Composizione di estratti dei lipidi polari e la composizione in acidi grassi di glycerolipids individuali sono determinati in un esperimento lipidi semplice e robusto profilo. A tal fine, glycerolipids sono isolati per cromatografia su strato sottile e sottoposti a transmetilazione dei loro gruppi acilici. Grassi methylesters acil sono quantificati da gas-cromatografia in fase liquida.

Video: Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

MALDI Sample Preparation: the Ultra Thin Layer Method

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

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

MALDI Preparazione del campione: il metodo ultra sottile strato

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

Ricerca

Biologia

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Isolamento di protoplasti da tessuti di 14-giorni di età piantine di Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Profiling anticorpi Systems Immunoprecipitazione luciferasi (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolamento di Labile Multi-proteina complessi da In vivo Controllato Cellular Cross-Linking e Immuno-magnetica Cromatografia di affinità

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floreale-dip Trasformazione Arabidopsis thaliana Per esaminare PTSO2:: β-glucuronidasi Reporter espressione genica

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Automated interazione idrofobica colonna di selezione cromatografia per l&#39;utilizzo in purificazione di proteine

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Isolamento efficiente e rapida di embrioni nelle prime fasi di<em&gt; Arabidopsis thaliana</em&gt; Semi

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

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 ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Colorazione istochimica di<em&gt; Arabidopsis thaliana</em&gt; Cellula secondaria Elementi da parete

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...