Ringraziamo Michael Reth, Hermann Schägger, e Margherita Camacho-Carvajal al sostegno scientifico. Questo lavoro è stato finanziato dalla FORSYS dal Bundesministerium für Bildung e Forschung (BMBF), dal BIOS dalla Deutsche Forschungsgemeinschaft (DFG), e sostenuta in parte dal Iniziativa eccellenza dei governi tedesco e Stato (GSC-4, Spemann Graduate School ).

<ol>
	<li>Arnold, I., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mitochondrial+F1F0-ATP+synthase+exists+as+a+dimer:+identification+of+three+dimer-specific+subunits.">Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.</a> EMBO J. 17 (24), 7170-7178 (1998).</li><li>Camacho-Carvajal, M. M., Wollscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+Blue+native/SDS+gel+electrophoresis+of+multi-protein+complexes+from+whole+cellular+lysates:+a+proteomics+approach.">Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach.</a> Mol. Cell. Proteomics. 3 (2), 176-182 (2004).</li><li>Heiss, K., Junkes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subproteomic+analysis+of+metal-interacting+proteins+in+human+B+cells.">Subproteomic analysis of metal-interacting proteins in human B cells.</a> Proteomics. 5 (14), 3614-3622 (2005).</li><li>Sali, A., Glaeser, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+words+to+literature+in+structural+proteomics.">From words to literature in structural proteomics.</a> Nature. 422, 216-225 (2003).</li><li>Schägger, H., Cramer, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217 (2), 220-230 (1994).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form.">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Anal. Biochem. 199 (2), 223-231 (1991).</li><li>Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylation+of+protein+complexes+may+lead+to+aggregation+as+well+as+to+loss+of+subunits+as+revealed+by+Blue+Native+PAGE.">Biotinylation of protein complexes may lead to aggregation as well as to loss of subunits as revealed by Blue Native PAGE.</a> J Immunol Methods. 252 (1-2), 171-174 (2001).</li><li>Schamel, W. W., Arechaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+multivalent+and+monovalent+TCRs+explains+high+sensitivity+and+wide+range+of+response.">Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response.</a> J. Exp. Med. 202, 493-503 (2005).</li><li>Schamel, W. W., Reth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomeric+and+oligomeric+complexes+of+the+B+cell+antigen+receptor.">Monomeric and oligomeric complexes of the B cell antigen receptor.</a> Immunity. 13 (1), 5-14 (2000).</li><li>Swamy, M., Minguet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+native+antibody-based+mobility-shift+technique+(NAMOS-assay)+to+determine+the+stoichiometry+of+multiprotein+complexes.">A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes.</a> J Immunol Methods. 324 (1-2), 74-83 (2007).</li><li>Swamy, M., Siegers, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+polyacrylamide+gel+electrophoresis+(BN-PAGE)+for+the+identification+and+analysis+of+multiprotein+complexes.">Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes.</a> Sci STKE. 2006 (345), 4-4 (2006).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+applications+of+blue-native+and+clear-native+electrophoresis.">Features and applications of blue-native and clear-native electrophoresis.</a> Proteomics. 8, 3974-3990 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

In questo studio, descriviamo l&#39;analisi di PPM da BN-PAGE. Un approccio 2D viene utilizzato al primo PPM separare in condizioni native, e poi ulteriormente suddividere nelle loro componenti singoli da una seconda dimensione SDS-PAGE. I campioni sono preparati con lisati cellulari. Per la solubilizzazione di PPM molti, un detergente appropriata è indispensabile, che conserva la struttura dei complessi proteici. Qui, usiamo 0,1% Triton X-100. Tuttavia, il detergente ottimale e la sua concentrazione adatta devono essere determinate empiricamente per ogni MPC. In caso di Triton X-100, per esempio, è stato riportato che le concentrazioni di detergenti a basso permettono l&#39;identificazione di una forma dimerica della F 1 F 0-ATPasi complesso (Arnold, Pfeiffer et al. 1998). Superiore Triton X-100 concentrazioni, tuttavia, portano alla dissociazione del dimero e ad un corrispondente aumento del monomerico F 1 F 0-ATPasi complesso. Ciò è in linea con uno dei nostri studi precedenti, sono stati dimostriamo che il polivalente cellule T complesso recettore (TCR) viene conservato quando estratto con basse concentrazioni di Brij 96, mentre l&#39;uso di una maggiore concentrazione o di un altro detergente chiamato digitonina risultati in l&#39;estrazione di monomerico TCR (Schamel, Arechaga et al. 2005). Detergenti comunemente usati che possono essere testati includono digitonina (da 0,5 a 1%), Triton X-100 (da 0,1 a 0,5%), Brij 96 (da 0,1 a 0,5%), o dodecylmaltoside (0,1 allo 0,5%). Questi reagenti sono detergenti non ionici, che tendono ad essere migliori per la stabilità MPC. Essere consapevoli del fatto che il contatto con SDS e altri detergenti aggressivi deve essere evitato (Camacho-Carvajal, Wollscheid et al. 2004). Dialisi del lisati è necessario per realizzare la separazione MPC in una BN-gel (Camacho-Carvajal, Wollscheid et al 2004.), (Heiss, Junkes et al 2005).. Sembra che la regolazione della concentrazione di sale o la rimozione delle impurità a basso peso molecolare è di fondamentale importanza per alta risoluzione. È interessante notare che anche le preparazioni di membrana e PPM, che sono stati immunopurified e poi eluito dalla anticorpi, sono adatti per BN-PAGE (Swamy, Siegers et al. 2006). In entrambi i casi, i campioni non devono essere dializzati per BN-PAGE la separazione, se lisi membrana o eluizione viene effettuata in BN-lisi buffer. Per la separazione delle proteine ​​da BN-PAGE, il colorante Coomassie blu è necessario, che si lega alle proteine ​​unspecifically e li copre con cariche negative. In tal modo, Coomassie blu consente la mobilità elettroforetica delle proteine ​​verso il catodo a pH neutro (Schägger e von Jagow 1991); (Schägger, Cramer et al 1994).. Inoltre, Coomassie blu impedisce l&#39;aggregazione delle proteine ​​nel gel di impilamento durante l&#39;elettroforesi. Per BN-PAGE, Coomassie G250 deve essere usato al posto di Coomassie R250 blu o blu Coomassie colloidale. Prima di eseguire una BN-gel, è necessario garantire che la percentuale di gel si adatta alla dimensione prevista del MPC di interesse. Prefabbricati BN-gel con pendenze diverse e tamponi adatti sono disponibili in commercio da Invitrogen (NativePAGE Novex Bis-Tris Gel System). Ma BN-gel può anche essere preparata usando un gradiente mixer insieme con una pompa persistaltic. Per garantire un gradiente intatto, il liquido deve fluire costantemente durante il getto e le bolle dovrebbero essere evitati. Si consiglia il caricamento di diverse diluizioni del campione sul gel perché sovraccarico può portare alla precipitazione delle proteine ​​durante il processo di elettroforesi. Inoltre, BN-gel dovrebbe essere eseguito a 4 ° C per evitare la degradazione delle proteine ​​e di mantenere intatto il PPM. Dopo BN-PAGE, la visualizzazione di MPC può essere raggiunto con la colorazione Coomassie blu brillante, colorazione argento o immunoblotting. Bande proteiche visualizzate mediante colorazione Coomassie o argento sono adatti per ulteriori analisi mediante spettrometria di massa (Camacho-Carvajal, Wollscheid et al. 2004). In caso di immunoblotting, le condizioni di trasferimento ottimale per i PPM di interesse devono essere determinati empiricamente. Essere consapevoli del fatto che Coomassie blu è anche trasferito durante assorbente di una BN-gel. Pertanto, il gel sarà incolore dopo il trasferimento di successo, mentre la membrana in grado di esercitare un colore blu. Inoltre, è importante ricordare che non tutti gli anticorpi primari, che lavora per il rilevamento dopo SDS-PAGE, è applicabile a immunoblotting su BN-PAGE. Può succedere che gli anticorpi non riconoscono il MPC di interesse perché la loro epitopo è nascosto nella conformazione nativa delle proteine. Per superare questo problema, è possibile denaturare le proteine ​​all&#39;interno della BN-gel prima del trasferimento facendo bollire il gel a breve nella tampone 1x campione SDS. Nel nostro esempio, noi non sottoporre il BN-gel direttamente al rilevamento di bande proteiche. Invece, abbiamo ulteriormente diviso il BN-PAGE-lisato separati da un secondo dimensisu SDS-PAGE. Nella seconda dimensione SDS-gel, proteine ​​monomeriche migrare all&#39;interno di un diagonale iperbolica a causa del gradiente di gel nel primo e il gel lineare nella seconda dimensione (Camacho-Carvajal, Wollscheid et al. 2004). Questo permette la facile identificazione dei PPM, dal momento che sono localizzati sotto questa diagonale iperbolica. Sottocomponenti di un distinto MPC sono separati in una linea verticale nella seconda dimensione SDS-PAGE. I componenti che rientrano nella composizione di PPM dinstinct diverse possono essere identificati su una linea orizzontale a seconda delle dimensioni del MPC. Tuttavia, è da considerare che gli spot di proteine ​​diverse che appare in una linea verticale potrebbe anche far parte di complessi separati che migrano nella stessa posizione in BN-PAGE. La prova finale che sono presenti nello stesso MPC può essere ottenuta con un anticorpo saggio basato su gel shift. In questo saggio, lisato cellulare viene incubato con un anticorpo contro una proteina rappresentata da uno dei luoghi individuati prima BN-PAGE. Ciò si traduce in uno spostamento di tutti i PPM che contengono questa proteina verso una massa molecolare maggiore nella prima dimensione. Altre proteine ​​che sono anche una parte di questi PPM subirà questo complesso specifico turno e sono quindi facili da identificare nella seconda dimensione SDS-gel. Non solo la composizione del MPC può essere analizzato BN-PAGE, ma anche la determinazione della loro stechiometria è possibile (e Schamel Reth 2000); (Schamel 2001), (Swamy, Minguet et al 2007).. A tal fine, un test Namos (nativo a base di anticorpi mobilità-shift test) può essere eseguita. Come nel anticorpi saggio basato su gel di turno, il lisati cellulari vengono incubati con subunità monoclonali specifici anticorpi. Questo porta a l&#39;induzione di immunoshifts elettroforetici nel BN-gel, che consentono l&#39;inferenza dalla misura dello spostamento sulla stechiometria di PPM In conlusion, BN-PAGE è adatto per l&#39;identificazione di PPM e la determinazione della loro dimensione, composizione, così come l&#39;abbondanza relativa. Eseguito come un test Namos, offre anche la possibilità di determinare la stechiometria di un certo MPC. Data la sua applicabilità generale, questa tecnica è uno strumento molto utile per la caratterizzazione di PPM (Dekker, Müller et al 1996.) (Wittig e Schagger 2008); (Wagner, Rehling et al 2009.) (Wittig e Schägger 2009) .

blue native polyacrylamide gel electrophoresis (bn-page) for analysis of multiprotein complexes from cellular lysates

Complessi multiproteici (PPM) svolgono un ruolo cruciale nella segnalazione cellulare, poiché la maggior parte delle proteine ​​si trovano in complessi funzionali o di regolamentazione con altre proteine ​​(Sali, Glaeser et al. 2003). Così, lo studio della proteina-proteina reti di interazione richiede la caratterizzazione dettagliata dei PPM di acquisire una comprensione integrativa della funzione della proteina e della regolamentazione. Per l&#39;identificazione e l&#39;analisi, MPC deve essere separata in condizioni native. In questo video, si descrive l&#39;analisi di PPM di blu elettroforesi nativa su gel di poliacrilamide (BN-PAGE). BN-PAGE è una tecnica che permette la separazione di PPM in una conformazione nativa con una risoluzione superiore a quella offerta da gel filtrazione o la densità ultracentrifugazione saccarosio, ed è quindi utile per determinare la dimensione del MPC, la composizione e l&#39;abbondanza relativa (Schägger e von Jagow 1991) ; (Schägger, Cramer et al 1994).. Con questo metodo, le proteine ​​sono separate in base alle loro dimensioni e la forma idrodinamica in una matrice di poliacrilamide. Qui, dimostriamo l&#39;analisi di PPM del totale lisati cellulari, sottolineando che la dialisi lisato è il passo fondamentale per rendere BN-PAGE applicabili a questi campioni biologici. Usando una combinazione di prima dimensione BN-e seconda dimensione SDS-PAGE, dimostriamo che PPM separati da BN-PAGE può essere ulteriormente suddivisa in loro singoli costituenti mediante SDS-PAGE. Visualizzazione dei componenti MPC su gel di separazione viene effettuata mediante immunoblotting standard. Come esempio di analisi da MPC BN-PAGE, abbiamo scelto la 19S ben caratterizzati eucariotiche, 20S, 26S e proteasomi.

Watch this Scientific Journal Video about Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates at JoVE.com

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In questo video, si descrive la caratterizzazione di complessi multiproteici (PPM) di blu elettroforesi su gel di poliacrilammide nativo (BN-PAGE). In una prima dimensione, dializzati lisati cellulari sono separati da BN-PAGE per identificare i singoli PPM. In una seconda dimensione SDS-PAGE, PPM di interesse sono ulteriormente suddivisi per analizzare i loro elettori mediante immunoblotting.

Blu nativi poliacrilammide gel elettroforesi (BN-PAGE) per l&#39;analisi di complessi multiproteici da lisati cellulari

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

blue-native-polyacrylamide-gel-electrophoresis-bn-page-for-analysis

Research

JoVE Journal

Biology

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

130.3K Views.  University of Freiburg. In questo video, si descrive la caratterizzazione di complessi multiproteici (PPM) di blu elettroforesi su gel di poliacrilammide nativo (BN-PAGE). In una prima dimensione, dializzati lisati cellulari sono separati da BN-PAGE per identificare i singoli PPM. In una seconda dimensione SDS-PAGE, PPM di interesse sono ulteriormente suddivisi per analizzare i loro elettori mediante immunoblotting.

Video: Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Denaturazione gel elettroforesi gradiente (DGGE)

Ricerca

Biologia

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Lo screening per aggregazione amiloide di semi-denaturazione detergente-Agarosio elettroforesi su gel

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Denaturazione Urea elettroforesi su gel poliacrilammide (PAG Urea)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Elettroforesi su gel agarosio per la separazione di frammenti di DNA

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Un nuovo approccio per l&#39;analisi comparativa dei multiproteico Complessi Basato su<sup&gt; 15</sup&gt; N metabolica Etichettatura e quantitativa Spettrometria di Massa

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Mapping basato AFM delle proprietà elastiche delle pareti cellulari: al Tissue, cellulare, e le risoluzioni subcellulari

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucina Agarosio elettroforesi su gel: Western Blotting per glicoproteine ​​ad alto peso molecolare


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Purificazione di Native Complessi di studio strutturale utilizzando un metodo Tag Tandem Affinity

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Analisi dei complessi della catena respiratoria mitocondriale in cellule umane coltivate mediante elettroforesi in Gel di poliacrilammide nativo blu e Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...