Nos gustaría agradecer la asistencia técnica de Inés Racke.

Parte 1: Preparación de la solución de tinción CBB <ol><li> 60-80 mg de CBB G-250 se disuelven en 1 litro de agua bidestilada por agitación durante 2-4 horas. Por último, 3 ml de HCl concentrado, se añade a la solución de color azul oscuro con agitación durante un minuto y se almacena en la oscuridad para su uso posterior. La solución se puede almacenar durante varias semanas hasta varios meses sin perder su eficacia manchas. </li><li> HCl concentrado debe ser manejado con el cuidado habitual und usada bajo una campana de humos. La solución final será aproximadamente a pH 2, por lo que se debe utilizar guantes y cualquier contacto con la piel debe ser evitado. </li></ol> Parte 2: SDS-PAGE <ol><li> Alícuotas apropiadas de muestras de proteínas se mezclan con tampón de carga a una concentración final de tampón de carga 1x. Usamos tampón de carga 2x 125mm Tris/H3PO4 (pH 7,5 a 25 ° C), 2 mM EDTA, SDS al 4%, DTT 200 mM, azul de bromofenol 0,02% y el 50% de glicerol. Otros tampones de carga para SDS-PAGE se puede usar también. </li><li> Las muestras de proteínas se calientan durante unos 5 minutos antes de la carga. Mientras tanto, la cámara de electroforesis en gel se prepara para la carrera. Usamos NuPAGE prefabricados 12.04% © Bis-Tris geles (Invitrogen) en un SureLock XCell ® Mini-Cell (Invitrogen), con economía de mercado-buffer como el funcionamiento de amortiguación, pero cualquier otro gel y el sistema de electroforesis se puede utilizar también. </li><li> Las muestras de proteínas se cargan en el gel y la electroforesis en una duración de 50 minutos a 220V. </li></ol> Parte 3: La tinción del gel <ol><li> El gel cassette está desmontada y el gel en una caja para los pasos posteriores de lavado. </li><li> Cerca de 100 ml de agua bidestilada se añade el gel y se calienta en el microondas durante 30 segundos. Calefacción debe detenerse antes de la ebullición se produce. La caja con el gel se coloca en un agitador durante 3-5 min. y este paso de lavado se repite dos veces con agua fresca. </li><li> Suficiente solución de tinción CBB se añade a cubrir el gel en la caja y la caja se calienta en el microondas durante 10 segundos. sin hervir. La caja con el gel se coloca en un agitador para el acabado de la tinción. Ya después de 1 minuto, las bandas de proteínas se puede observar, después de 15-30 min. la tinción es lo suficientemente fuerte como en la mayoría de los casos. </li><li> La solución colorante se escurre y 50-100 ml de agua bidestilada, se añade con el fin de destain aún más la luz de fondo azul del gel en un agitador. El agua puede ser sustituida por agua dulce para la decoloración más si es necesario. </li><li> El gel puede ser escaneado, fotografiado o secas para almacenamiento a largo plazo </li></ol> Parte 4: Resultados del representante: Ver fig. 1 para un gel teñido correctamente siguiendo el procedimiento descrito. Ver fig. 2 para un gel que no se ha lavado el tiempo suficiente antes de la tinción y coloración residual SDS inhibe eficiente. Tenga en cuenta que los carriles marcador (*) contienen la misma cantidad de proteínas marcadoras. <img src="/files/ftp_upload/1350/Fig1.png" alt="" /> Fig. 1: Representante manchadas gel después de cargar las muestras de una purificación de proteínas (*: marcador de peso molecular). <img src="/files/ftp_upload/1350/Fig2.png" alt="" /> Fig. 2: CBB gel de colores que no se ha lavado el tiempo suficiente antes de la tinción CBB. Las bandas de proteínas aparecen más débiles (nótese que el carril * marcador contiene la misma cantidad de proteínas como en la fig. 1).

<ol>
	<li>Wondrak, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+for+fast+visualization+of+protein.">Process for fast visualization of protein.</a> US patent. , (2001).</li><li>Wondrak, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+for+fast+visualization+of+protein.">Solution for fast visualization of protein.</a> US patent. , (2001).</li></ol>

No hay conflicto de intereses. El procedimiento descrito anteriormente fue publicado originalmente en la solicitud de patente por EM Wondrak (ver ref.).

<ul><li> Los pasos de lavado son fundamentales para la tinción eficiente de las proteínas. Una reducción del tiempo de lavado por debajo de 2 min o disminución del volumen de agua (&lt;50 ml) puede dar lugar a bandas de proteínas pálido azul, muy probablemente debido a una mayor cantidad de residuos SDS en el gel. </li><li> Si las proteínas van a ser analizados por espectrometría de masas, los pasos de calentamiento en el horno de microondas debe ser omitido, el tiempo para el lavado del gel extendido a unos 10 minutos en cada paso y el...

<table><tbody><tr><td>Coomassie Brilliant Blue G-250</td><td>AppliChem</td><td>A3480</td><td>any other CBB G-250 could be used as well</td></tr><tr><td>Concentrated HCl</td><td></td><td></td><td></td></tr></tbody></table>

staining of proteins in gels with coomassie g-250 without organic solvent and acetic acid

En los protocolos clásicos de proteínas tinción con Coomassie Brilliant Blue (CBB), las soluciones con un alto contenido de disolventes orgánicos tóxicos e inflamables (metanol, etanol o 2-propanol) y ácido acético se utilizan para la fijación, tinción y decoloración de las proteínas en un gel de SDS después -PAGE. Para acelerar el procedimiento, el calentamiento de la solución de tinción en el horno de microondas por un corto período de tiempo se utiliza con frecuencia. Esto usualmente resulta en la evaporación de metanol tóxicos o peligrosos, etanol o 2-propanol y un fuerte olor a ácido acético en el laboratorio que deben evitarse por motivos de seguridad. En un protocolo publicado originalmente en dos solicitudes de patente por EM Wondrak (US2001046709 (A1), US6319720 (B1)), una composición alternativa de la solución de tinción se describe en la que no solvente orgánico o ácido se utiliza. El CBB se disuelve en agua bidestilada (60-80mg de CBB G-250 por litro) y 35 mM HCl se agrega como el único compuesto otros en la solución de tinción. El CBB staning del gel se realiza después de SDS-PAGE y minuciosamente del gel en agua bidestilada. Al calentar el gel durante el lavado y la tinción de pasos, el proceso puede terminar más rápido y no compunds tóxicos o peligrosos se evapora. La tinción de las proteínas ocurre ya dentro de 1 minuto después de calentar el gel en solución de tinción y se desarrolla completamente después de 15-30 minutos con un fondo azul que es un poco desteñido por completo por el lavado prolongado del gel teñido con agua bidestilada, sin afectar a la proteína manchado bandas.

Watch this Scientific Journal Video about Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid at JoVE.com

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

Un protocolo corto para la tinción de proteínas con Coomassie Brilliant Blue (CBB) G-250 en geles de poliacrilamida se describe sin el uso de disolventes orgánicos o en ácido acético como en los procedimientos clásicas de tinción con CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

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

staining-proteins-gels-with-coomassie-g-250-without-organic-solvent

Research

JoVE Journal

Biology

53.1K vistas.  EMBL Heidelberg. Un protocolo corto para la tinción de proteínas con Coomassie Brilliant Blue (CBB) G-250 en geles de poliacrilamida se describe sin el uso de disolventes orgánicos o en ácido acético como en los procedimientos clásicas de tinción con CBB.

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

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Mejora En-gel β-eliminación para ligada al O y exhaustiva sulfo-glycomics reductoras de Espectrometría de Masas

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Investigación

Química

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Electroforesis en Gel bidimensional juntada con métodos de espectrometría de masas para el análisis del proteoma de tejido humano Adenoma pituitario

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Investigación sobre el cáncer

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Tinción de proteínas en geles de

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Biología

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Tinción fluorescente de plata de proteínas en geles de poliacrilamida

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Bioquímica

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Identificación de fosfato de inositol o proteínas que interactúan con fosfoinositida por cromatografía de afinidad acoplada a Western Blot o espectrometría de masas

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Rápido y sensible coloidal Coomassie G-250 La tinción de proteínas en geles de poliacrilamida

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.

Extracción y visualización de agregados proteicos después del tratamiento de Escherichia coli con un estresor proteotóxico

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

Fraccionamiento mitocondrial en muestras de tejido pequeño para análisis supercomplejo

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Autor destacado: Desvelando la dinámica del sistema de fosforilación oxidativa y las funciones mitocondriales en la salud y la enfermedad

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Aislamiento de mitocondrias para el análisis de supercomplejos mitocondriales a partir de muestras de pequeños tejidos y cultivos celulares 

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

Resumen

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Aislamiento de mitocondrias para el análisis de supercomplejos mitocondriales a partir de muestras de pequeños tejidos y cultivos celulares

Aislamiento de mitocondrias para el análisis de supercomplejos mitocondriales a partir de muestras de pequeños tejidos y cultivos celulares