Ringraziamo Oliver Brüstle e tutti i membri del Gruppo Engineering Cellule Staminali, Università di Bonn, per il supporto e discussioni importanti. Ringraziamo Sabine Schenk per la preparazione di SDS-PAGE e supporto duraturo in tutto il progetto. Nicole Russ e Magerhans Anna fornito un eccellente supporto tecnico. Inoltre, vorremmo ringraziare Andreas Bär e Mertens Sheila per la produzione del film. Questo lavoro è stato supportato anche da finanziamenti della Fondazione Volkswagen (Az I/77864) e dal Ministero tedesco dell&#39;Istruzione e della Ricerca (BMBF, 01 GN 0813).

Costruzione del vettore di espressione e di espressione: Il pSESAME Cre-vettore di espressione è stato costruito inserendo un frammento di Cre-codifica in pSESAME tramite siti di restrizione AvrII e NheI utilizzando metodi standard di clonazione. pSESAME codifica per una proteina di fusione costituita da una istidina-tag, TAT-dominio, la sequenza NLS e Cre, abbreviato HTNCre. A manifestare HTNCre la pSESAME-Cre è stato trasformato in TUNER (DE3) pLacI e utilizzato per preparare uno stock di glicerolo. <ol><li> Un over-notte della cultura è stato inoculato con una punta di pipetta rivestito con batteri trasformati dallo stock glicerolo. La cultura durante la notte consisteva di LB mezzi di comunicazione integrata con 0,5% di glucosio [v / v] e carbenicillina ad una concentrazione finale di 50 mg / ml ed è stato permesso di crescere a 37 ° C per 16 ore. </li><li> Il giorno dopo la densamente cresciuto durante la notte della cultura è stata utilizzata per inoculare la cultura espressione di un rapporto di 1 a 40 e è stato messo in incubatrice a 37 ° C. Cultura espressione consisteva di mezzi TB integrata con 0,5% di glucosio [v / v] e ampicillina ad una concentrazione finale di 100 mg / ml. </li><li> Ad un OD 595 di 1,5 cultura espressione è stata indotta con IPTG 0,5 mm per 1 h. </li><li> Successivamente i batteri sono stati raccolti per centrifugazione a 5000 rpm per 10 minuti in un rotore SLA3000. </li><li> Pellet di batteri sono stati conservati a meno 20 ° C fino alla purificazione. </li></ol> Purificazione di cellule permeabili proteine: <ol><li> Pellet congelato i batteri sono stati risospesi in 10 ml di tampone di lisi per la cultura fiasco litro per 15 minuti a temperatura ambiente. </li><li> Sospensione è stata poi incubate con 1 mg / ml di lisozima per altri 15 minuti, mescolando a temperatura ambiente. </li><li> 25 U / mL benzonasi è stato aggiunto in seguito durante la miscelazione e incubate per 15 minuti a temperatura ambiente. </li><li> Dopo sonicazione in ghiaccio per 1,5 minuti con 0,5 s impulsi al 45% della potenza, 1 mL di tampone a freddo sale tartarico (TSB) per mL sospensione è stata accuratamente aggiunto durante la miscelazione e incubate per 5 minuti sul ghiaccio. SDS-PAGE campione della frazione lisato (L) è stata presa. </li><li> Lisato eliminato è stato ottenuto per centrifugazione a 4 ° C per 30 minuti a 30.000 g. SDS-PAGE di campioni delle frazioni solubili (S) e insolubili (I) sono state prese. </li><li> Il surnatante è stato trasferito in fresco 50 ml tubi falco e ° C con 2 ml di 50% Ni-NTA liquami per ogni litro di cultura espressione iniziale. Fu poi delicatamente mescolata per 1 ora a 4 </li><li> La sospensione è confezionato in una colonna di flusso per gravità EconoPac (SDS-PAGE campione di flow-through frazione (FT) è stata scattata) e lavato due volte con 5 posti letto-volumi di tampone di lavaggio. SDS-PAGE di campioni di entrambe le frazioni di lavaggio (W1 &amp; W2) sono stati raccolti. </li><li> HTNCre contenenti frazioni sono stati eluiti con 3 letto volumi di tampone di eluizione del campione e della frazione di eluato (E) per SDS-PAGE analisi è stata presa. </li><li> Imidazolo è stato rimosso dalla dialisi frazione contro il buffer di eluizione di sale due volte. </li><li> La soluzione proteica è stata ulteriormente concentrata da dialisi contro tampone glicerolo due volte. In tutte le fasi dialisi il rapporto del buffer da campione è stato di almeno 50. Questa procedura ha portato a una soluzione madre contenente HTNCre glicerolo ad una concentrazione al solito tra i 200 e 450 micron, cioè 1 litro di cultura espressione si tradurrà in ~ 12 mg di proteina. Esempio di magazzino glicerolo (GS) per SDS-PAGE analisi sono stati raccolti. Soluzione madre HTNCre può essere conservata a meno 20 ° C. </li></ol><img border="0" src="/files/ftp_upload/1627/1627fig1tmb.jpg" alt="Figura 1" /> Figura 1: SDS-PAGE analisi dei campioni raccolti durante il processo di purificazione della Cre ricombinasi. Induzione di espressione Cre è indicato da banda dominante in frazione lisato. Anche se una parte della proteina è insolubile la proteina Cre può essere ulteriormente arricchito, come visto in eluato e frazioni magazzino glicerolo. L: lisato, I: Insolubile, S: supernatante, FT: flow-through, W: lavaggio, E: eluato, GS: Stock Gylcerol. Si prega di <a href="https://www.jove.com/files/ftp_upload/1627/1627fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1. Trasduzione di proteine ​​in staminali embrionali murine (ES), le cellule: <ol><li> Cellule ES portando un condizionale β-galattosidasi giornalista 5 costruire sono state seminate come singole cellule utilizzando TrypLE ™ Express per la dissociazione di cellule aderenti. Dopo 4 a 6 ore le cellule avevano nuovamente attaccati e il mezzo è stato rimosso. </li><li> Successivamente le cellule ES sono state incubate con HTNCre contenenti media per 16 ore. <ul><li> Una quantità adeguata di proteine ​​HTNCre (corrispondenti a 10 micron) fuori dello stock glicerolo è stato diluito in media ES e successivamente filtrato sterile (0,22 micron). </li></ul></li><li> Dopo trasduzione medio di proteine ​​è stato cambiato di nuovo a mezzo di crescita normale. </li><li> Dopo due giorni le cellule sono state lavate con PBS e fissate con 4% paraformaldeide (PFA) per 10 minuti. </li><li> Due ulteriorizionale fasi di lavaggio con PBS sono stati eseguiti prima di X-Gal colorazione è stata eseguita. </li><li> Cellule fissate sono state coperte con uno strato di X-Gal soluzione colorante 6 e incubate per tutta la notte a 37 ° C. </li></ol> Rappresentante dei risultati: Giorno dopo X-Gal è stato aspirato soluzione colorante e le cellule sono state coperte da uno strato di PBS per l&#39;analisi di microscopia. Dall&#39;80 al 100% delle cellule ricombinano potrebbe essere osservate entro le cellule staminali embrionali murine giudicati da β-galattosidasi.

<ol>
	<li>Gump, J. M., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAT+transduction:+the+molecular+mechanism+and+therapeutic+prospects.">TAT transduction: the molecular mechanism and therapeutic prospects.</a> Trends Mol Med. 13 (10), 443-448 (2007).</li><li>Edenhofer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transduction+revisited:+novel+insights+into+the+mechanism+underlying+intracellular+delivery+of+proteins.">Protein transduction revisited: novel insights into the mechanism underlying intracellular delivery of proteins.</a> Curr Pharm Des. 14 (34), 3628-3636 (2008).</li><li>Branda, C. S., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Talking+about+a+revolution:+The+impact+of+site-specific+recombinases+on+genetic+analyses+in+mice.">Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice.</a> Dev Cell. 6 (1), 7-28 (2004).</li><li>Nolden, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+recombination+in+human+embryonic+stem+cells+induced+by+cell-permeant+Cre+recombinase.">Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase.</a> Nat Methods. 3 (6), 461-467 (2006).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+site-directed+recombination+in+mouse+embryonic+stem+cells.">Inducible site-directed recombination in mouse embryonic stem cells.</a> Nucleic Acids Res. 24 (4), 543-548 (1996).</li><li>Peitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purification+of+cell-permeant+Cre+and+germline+transmission+after+transduction+into+mouse+embryonic+stem+cells.">Enhanced purification of cell-permeant Cre and germline transmission after transduction into mouse embryonic stem cells.</a> Genesis. 45 (8), 508-517 (2007).</li><li>Bosnali, M., Edenhofer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transducible+versions+of+transcription+factors+Oct4+and+Sox2.">Generation of transducible versions of transcription factors Oct4 and Sox2.</a> Biol Chem. 389 (7), 851-861 (2008).</li><li>Landry, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx+genes+are+direct+targets+of+Scl/Tal1+in+the+yolk+sac+and+fetal+liver.">Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver.</a> Blood. 111 (6), 3005-3014 (2008).</li></ol>

Durante il processo di purificazione della proteina di fusione Cre è importante non tralasciare l&#39;aggiunta di buffer di ghiaccio freddo TBS prima centrifugazione. Altrimenti Cre ricombinasi tende a precipitare all&#39;interno del buffer glicerolo. Se la frazione eluato sembra diventare torbido a causa della concentrazione di buffer di eluizione delle proteine ​​di fusione, aggiungere fino a quando la soluzione ha eliminato di nuovo. L&#39;applicazione di 10 mM di proteina di fusione Cre risultati in genere in un efficienza ricombinazione tra 80 e 100%. Di siero fetale bovino (FCS) è un componente importante della media delle cellule ES inibisce fortemente trasduzione delle proteine. Pertanto alta concentrazione di Cre ricombinasi doveva essere usato. Quando si lavora nel siero senza condizioni meno proteine ​​(0,5-2 mM) può essere utilizzato per ottenere efficienze ricombinazione simili. Con il sistema vettoriale pSESAME a portata di mano si può applicare la tecnica di trasduzione di proteine ​​ad altre proteine ​​tra cui fattori di trascrizione come Oct4 e Sox2 Scl/Tal1 7 e 8.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>TUNER (DE3) pLacI</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70625</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3783.2</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>T876.1</td>
<td>&nbsp;</td>
<tr>
<td>Trizma Base</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>HCl</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>4625.1</td>
<td>&nbsp;</td>
<tr>
<td>Imidazol</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>X998.4</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>9265.2</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>2363.4</td>
<td>&nbsp;</td>
<tr>
<td>Trypton/Pepton</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>8952.4</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4 </td>
<td>&nbsp;</td>
<td>Roth</td>
<td>P749.2</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>3904.1</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9518</td>
<td>&nbsp;</td>
<tr>
<td>Carbenicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6344.2</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-Tartaric acid, disodium salt </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50% Ni-NTA slurry</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>R901-15</td>
<td>&nbsp;</td>
<tr>
<td>EconoPac columns</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>732-1010</td>
<td>&nbsp;</td>
<tr>
<td>Sterile filter 0,22&mu;m</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (PFA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Yeast extract, Trypton/Pepton, NaCl</td>
</tr>
<tr>
<td>TB medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Yeast extract, Trypton/Pepton, Glycerol, K2HPO4, KH2PO4</td>
</tr>
<tr>
<td>Lysis Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50 mM Na2HPO4, 5 mM Tris, pH 7.8</td>
</tr>
<tr>
<td>Tartaric Salt Buffer (TSB)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM Imidazol</td>
</tr>
<tr>
<td>Washing Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB, 500 mM NaCl, 15 mM Imidazol</td>
</tr>
<tr>
<td>Elution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB, 500 mM NaCl, 250 mM Imidazol</td>
</tr>
<tr>
<td>High Salt Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>600 mM NaCl, 20 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>Gylcerol Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50% glycerol, 500 mM NaCl, 20 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>TrypLE&trade; Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ESGRO (LIF)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NEAA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11140035</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030024</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>31350010</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960044</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>PAA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal staining solution:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4 mM K3(FeIII(CN)6), 4 mM K4(FeII(CN)6), 2mM MgCl2 0.4 mg/mL X-Gal solved in PBS</td>
</tr>
<tr>
<td>K3(FeIII(CN)6)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3367</td>
<td>&nbsp;</td>
<tr>
<td>K4 (FeII(CN)6)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-9387</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4252</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

engineering cell-permeable protein

La tecnica di trasduzione proteina consente la fornitura diretta di materiale biologicamente attivo nelle cellule dei mammiferi [per una rassegna vedi 1,2]. Per questo si può fare uso delle capacità translocating dei cosiddetti peptidi cellula penetrando (CPP), designato anche come domini di trasduzione delle proteine ​​(PTDs). La TAT-CPP derivati ​​dal virus dell&#39;immunodeficienza umana di tipo 1 (HIV-1) Tat (trans-attivatore della trascrizione), proteina è stato ampiamente utilizzato. La carica positiva TAT promuove la permeabilità cellulare superando così le barriere della membrana cellulare per endocitosi o / e la penetrazione diretta della membrana 2. In combinazione con un segnale di localizzazione nucleare (NLS), proteine ​​di fusione sono in grado di entrare nel nucleo funzionalità espositiva. La nostra presentazione video dimostra, come esemplificazione per l&#39;ingegneria delle cellule permeabili le proteine, la costruzione, la produzione e l&#39;applicazione di una cellula-permeabile versione del DNA modificanti enzima Cre. Cre è una ricombinasi sito-specifiche in grado di riconoscere e ricombinare base di 34 siti loxP coppia in cellule di mammifero in vitro e in vivo. Pertanto, il Cre / loxP sistema è ampiamente utilizzato per indurre condizionalmente mutazioni nel genoma delle cellule viventi 3,4. La consegna dei attivo Cre ricombinasi alle cellule, tuttavia, rappresenta una limitazione. Noi descriviamo il sistema vettore pSESAME, che permette un inserimento diretto del gene d&#39;interesse e offre una piattaforma per clonare rapidamente domini diversi e tag utilizzati all&#39;interno del vettore in maniera comoda e standardizzata. Riorganizzazione dei tag diversi ha dimostrato di modificare le proprietà biochimiche delle proteine ​​di fusione che fornisce la possibilità di ottenere una maggiore resa e una migliore solubilità. Dimostriamo come esprimere e purificare cellule ricombinanti permeanti proteine ​​e da E. coli. La funzionalità della proteina ricombinante Cre è finalmente convalidato nelle cellule in coltura per valutare la sua attività intracellulare ricombinasi.

Watch this Scientific Journal Video about Engineering Cell-permeable Protein at JoVE.com

Engineering Cell-permeable Protein

Trasduzione proteina consente la fornitura diretta di proteine ​​biologicamente attive nelle cellule. A differenza dei metodi tradizionali come la transfezione di DNA o di trasduzione virale questo non invasivo consente paradigma altamente efficiente manipolazione cellulare in modo titolabile eludere tossicità cellulare e il rischio di trasformazione oncogena permanente da modificazione genetica.

Ingegneria Cell-permeabile proteine

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Research

JoVE Journal

Biology

14.3K Views.  University of Bonn - Life & Brain Center and Hertie Foundation. Trasduzione proteina consente la fornitura diretta di proteine ​​biologicamente attive nelle cellule. A differenza dei metodi tradizionali come la transfezione di DNA o di trasduzione virale questo non invasivo consente paradigma altamente efficiente manipolazione cellulare in modo titolabile eludere tossicità cellulare e il rischio di trasformazione oncogena permanente da modificazione genetica.

Video: Engineering Cell-permeable Protein

Experimental Approaches to Tissue Engineering

Approcci sperimentali di ingegneria dei tessuti

Ricerca

Biologia

Micro-scale Engineering for Cell Biology

Micro-scala di Ingegneria di biologia cellulare

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Incapsulamento cellulare in idrogel 3D per l&#39;ingegneria tissutale

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

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

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

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 ...

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 ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

Proteine ​​saggezza: un banco di lavoro per<em&gt; In silico</em&gt;<em&gt; De novo</em&gt; Progettazione di biomolecole

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Genoma Ingegneria mouse usando Nucleases Designer

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<em&gt; Escherichia coli</em

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

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Diretto Protein Consegna a cellule di mammifero Utilizzando Cell-permeabile Cys<sub&gt; 2</sub&gt; -Il suo<sub&gt; 2</sub&gt; domini zinc-finger

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...