Damos las gracias a Michael Reth, Schagger Hermann, y Margarita Camacho-Carvajal de apoyo científico. Este trabajo fue financiado por FORSYS de la Bildung Bundesministerium fuer Forschung y de Alemania (BMBF), por el BIOS de la Deutsche Forschungsgemeinschaft (DFG), y apoyado en parte por la Iniciativa de Excelencia de los Gobiernos de Alemania Federal y el Estado (GSC-4, Spemann Escuela de Posgrado ).

<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>

En este estudio, se describe el análisis de los países socios mediterráneos por BN-PAGE. Un enfoque en 2D se utiliza para separar los PSM primera virtud de las condiciones, y luego más que dividir en sus componentes individuales de una segunda dimensión SDS-PAGE. Las muestras se preparan a partir de lisados ​​celulares. Para la solubilización de muchos países socios mediterráneos, un detergente apropiado que se necesita, que conserva la estructura de los complejos de proteínas. Aquí, nosotros usamos un 0,1% Triton X-100. Sin embargo, el detergente óptimo y su concentración adecuada tiene que ser determinada empíricamente para cada MPC. En el caso de Triton X-100, por ejemplo, se ha informado de que las bajas concentraciones de detergente permite la identificación de una forma dimérica de la F 1 F 0-ATPasa complejos (Arnold, Pfeiffer et al. 1998). Mayor Triton X-100 concentraciones, sin embargo, a la disociación del dímero y el correspondiente aumento de las monoméricas F 1 F 0-ATPasa complejo. Esto está en consonancia con uno de nuestros estudios anteriores, se nos muestran que el polivalente de células T complejo receptor (TCR) se conserva cuando se extrae con bajas concentraciones de Brij 96, mientras que el uso de una mayor concentración o de otro detergente llamado digitonina resultados en la extracción de monómero TCR (Schamel, Arechaga y cols. 2005). Detergentes de uso común que pueden ser probados incluyen digitonina (0,5 a 1%), Triton X-100 (0,1 a 0,5%), Brij 96 (de 0,1 a 0,5%), o dodecylmaltoside (0,1 a 0,5%). Estos reactivos son los detergentes no iónicos, que tienden a ser mejor para la estabilidad de MPC. Tenga en cuenta que el contacto con SDS y otros detergentes fuertes se debe evitar (Camacho-Carvajal, Wollscheid et al. 2004). Diálisis de los lisados ​​se requiere para lograr la separación MPC en un BN-gel (Camacho-Carvajal, Wollscheid et al 2004).; (Heiss, Junkes et al 2005).. Parece que el ajuste de la concentración de sal o la eliminación de las impurezas de bajo peso molecular es crucial para alta resolución. Es de destacar que también las preparaciones de membrana y los PSM, que han sido inmunopurificados y posteriormente eluido de los anticuerpos, son adecuados para BN-PAGE (Swamy, Siegers et al. 2006). En ambos casos, las muestras no tienen que ser dializados para la separación de BN-PAGE, si la lisis de membrana o de elución se lleva a cabo en tampón BN-lisis. Para la separación de proteínas por BN-PAGE, el colorante azul de Coomassie es necesario, que se une inespecíficamente a las proteínas y los cubre con las cargas negativas. De esta manera, azul de Coomassie permite la movilidad electroforética de las proteínas hacia el cátodo a un pH neutro (Schagger y von Jagow 1991); (Schagger, Cramer et al, 1994).. Además, azul de Coomassie impide la agregación de proteínas en el gel de apilamiento durante la electroforesis. BN-PAGE, Coomassie G250 tiene que ser usado en lugar de azul de Coomassie R250 o coloidal azul Coomassie. Antes de ejecutar un BN-gel, es necesario garantizar que el porcentaje del gel se adapta al tamaño esperado de la MPC de interés. Prefabricados BN-geles con gradientes de diferentes tampones adecuados y están disponibles comercialmente a partir de Invitrogen (NativePAGE Novex Bis-Tris Gel System). Sin embargo, BN-geles también pueden prepararse usando un mezclador de gradiente, junto con una bomba persistaltic. Para garantizar un gradiente intacta, el líquido debe fluir constantemente durante el vaciado y las burbujas se debe evitar. Recomendamos la carga de las diluciones de muestras diferentes en el gel, porque la sobrecarga puede dar lugar a la precipitación de proteínas durante el proceso de electroforesis. Además, el BN-geles se debe ejecutar a 4 ° C para evitar la degradación de proteínas y para mantener el PSM intacto. Después de BN-PAGE, la visualización de los PSM se puede lograr mediante la tinción de azul brillante de Coomassie, tinción de plata o inmunotransferencia. Las bandas de proteínas visualizados por tinción con Coomassie o plata son adecuados para su posterior análisis por espectrometría de masas (Camacho-Carvajal, Wollscheid et al. 2004). En el caso de inmunotransferencia, las condiciones de transferencia óptima de los países socios mediterráneos de interés tiene que ser determinada empíricamente. Tenga en cuenta que Coomassie azul también es transferido durante el secante de un BN-gel. Por lo tanto, el gel se incoloro después de la transferencia con éxito, mientras que la membrana va a ejercer un color azul. Además, es importante mencionar que no todos los anticuerpos primarios, que trabaja en la detección después de SDS-PAGE, es aplicable a inmunotransferencia en BN-PAGE. Puede ocurrir que los anticuerpos no reconocen la MPC de interés debido a su epítopo se oculta en la conformación nativa de las proteínas. Para superar este problema, es posible para desnaturalizar las proteínas dentro de la BN-gel antes de la transferencia al hervir el gel en tampón 1x breve muestra de SDS. En nuestro ejemplo, que no tema el BN-gel directamente a la detección de bandas de proteínas. En su lugar, divide a la BN-PAGE de lisado separados por un segundo dimensien SDS-PAGE. En la segunda dimensión SDS-gel, proteínas monoméricas migrar dentro de una diagonal hiperbólico, debido a que el gel de gradiente en el primero y el gel lineal en la segunda dimensión (Camacho-Carvajal, Wollscheid et al. 2004). Esto permite la fácil identificación de los países socios mediterráneos, ya que se localizan por debajo de esta hiperbólica diagonal. Subcomponentes de una distinta MPC se separan en una línea vertical en la segunda dimensión SDS-PAGE. Componentes que son constituyentes de los PSM dinstinct varios se pueden identificar en una línea horizontal en función del tamaño de la MPC. Sin embargo, hay que tener en cuenta que varios puntos de proteínas que aparecen en una línea vertical también podría formar parte de los complejos que migran por separado en la misma posición en BN-PAGE. La prueba final de que se encuentran en la misma MPC se puede obtener mediante un ensayo en gel a base de anticuerpos turno. En este ensayo, lisado celular se incuba con un anticuerpo contra una proteína representada por uno de los puntos identificados antes de la BN-PAGE. Esto se traduce en un cambio de todos los países socios mediterráneos que contienen esta proteína hacia una mayor masa molecular en la primera dimensión. Otras proteínas que son también una parte de estos países socios mediterráneos se someterán a este cambio de complejos específicos y por lo tanto fácil de identificar en la segunda dimensión SDS-gel. No sólo la composición de los países socios mediterráneos pueden ser analizados por BN-PAGE, sino también la determinación de su estequiometría es posible (y Schamel Reth 2000); (Schamel 2001), (Swamy, Minguet y otros, 2007.). Para ello, un ensayo de NAMOS (nativo basado en anticuerpos movilidad cambio de ensayo) se puede realizar. Al igual que en el ensayo de gel a base de anticuerpos cambio, los lisados ​​celulares se incuban con anticuerpos monoclonales subunidad anticuerpos específicos. Esto conduce a la inducción de immunoshifts electroforética en los geles BN-, que permiten la inferencia de la medida del cambio en la estequiometría de los países socios mediterráneos En conlusion, BN-PAGE es adecuado para la identificación de los países socios mediterráneos y la determinación de su tamaño, composición, así como la abundancia relativa. Realizado como un ensayo NAMOS, sino que también ofrece la posibilidad de determinar la estequiometría de una cierta MPC. Teniendo en cuenta su aplicabilidad general, esta técnica es una herramienta muy útil para la caracterización de los países socios mediterráneos (Dekker, Müller y otros, 1996.) (Wittig y Schagger 2008); (Wagner, Rehling y otros, 2009.) (Wittig y Schagger 2009) .

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

Complejos multiproteicos (PSM) juegan un papel crucial en la señalización celular, ya que la mayoría de las proteínas se pueden encontrar en complejos funcionales o de reglamentación con otras proteínas (Sali, Glaeser et al. 2003). Así, el estudio de las redes de interacción proteína-proteína requiere de la caracterización detallada de los países socios mediterráneos para obtener una comprensión integradora de la función de la proteína y la regulación. Para la identificación y el análisis, los países socios mediterráneos deben ser separados en condiciones nativas. En este video, se describe el análisis de los países socios mediterráneos por el azul de electroforesis en gel de poliacrilamida nativo (BN-PAGE). BN-PAGE es una técnica que permite la separación de los PSM en una conformación nativa con una resolución mayor que la ofrecida por filtración en gel o ultracentrifugación de densidad de sacarosa, y por tanto es útil para determinar el tamaño del MPC, la composición y abundancia relativa (Schagger y von Jagow 1991) ; (Schagger, Cramer et al, 1994).. Mediante este método, las proteínas se separan según su tamaño y forma hidrodinámica en una matriz de poliacrilamida. En este caso, se demuestra el análisis de los PSM del total de lisados ​​celulares, señalando que la diálisis lisado es el paso crucial para hacer BN-PAGE aplicable a las muestras biológicas. Usando una combinación de primera dimensión BN-y segunda dimensión SDS-PAGE, se muestra que los países socios mediterráneos separados por BN-PAGE se pueden subdividir en sus componentes individuales por SDS-PAGE. La visualización de los componentes MPC después de la separación de gel se realiza por immunoblotting estándar. A modo de ejemplo para el análisis de MPC por BN-PAGE, se optó por el 19S bien caracterizados eucariotas, 20S, 26S y los proteasomas.

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

En este video, se describe la caracterización de los complejos multiproteicos (PSM) por el azul electroforesis nativa en gel de poliacrilamida (BN-PAGE). En una primera dimensión, se dializan lisados ​​celulares están separados por BN-PAGE para identificar PSM individual. En una segunda dimensión SDS-PAGE, PSM de interés se subdividen para analizar sus componentes por inmunotransferencia.

Azul nativos electroforesis en gel de poliacrilamida (BN-PAGE) para el análisis de los complejos Multiproteinas de lisados ​​celulares

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 vistas.  University of Freiburg. En este video, se describe la caracterización de los complejos multiproteicos (PSM) por el azul electroforesis nativa en gel de poliacrilamida (BN-PAGE). En una primera dimensión, se dializan lisados ​​celulares están separados por BN-PAGE para identificar PSM individual. En una segunda dimensión SDS-PAGE, PSM de interés se subdividen para analizar sus componentes por inmunotransferencia.

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

Gradiente desnaturalizante electroforesis en gel (DGGE)

Investigación

Biología

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.

La detección de la agregación amiloide de Semi-desnaturalización electroforesis en gel de agarosa-detergente

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.

Poliacrilamida electroforesis en gel de urea (urea PÁGINA)

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.

Electroforesis en gel de agarosa para la separación de los fragmentos de ADN

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 nuevo enfoque para el análisis comparativo de Multiproteinas complejos basados ​​en<sup&gt; 15</sup&gt; N metabólico Etiquetado y cuantitativa de Espectrometría de Masas

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.

Cartográficas basadas en AFM de las propiedades elásticas de las paredes celulares: en tejido, celular, y las Resoluciones Subcelulares

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.

La mucina en gel de agarosa de electroforesis: Western Blotting para glicoproteínas de alta peso molecular


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.

Purificación de Complejos nativo para el estudio estructural se utiliza un método 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.

Análisis de complejos de cadena respiratoria mitocondrial en células humanas cultivadas usando electroforesis en Gel de poliacrilamida nativo azul e Immunoblotting

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

Azul nativos electroforesis en gel de poliacrilamida (BN-PAGE) para el análisis de los complejos Multiproteinas de lisados ​​celulares

Azul nativos electroforesis en gel de poliacrilamida (BN-PAGE) para el análisis de los complejos Multiproteinas de lisados celulares