Este trabajo está dedicado a la memoria del fallecido Dr. Zvi Selinger, que fue sede de la investigación original se describe aquí.

Linealización de la curva de calibración Bradford Proteínas: El Coomassie brillante ensayo de la proteína azul, conocido comúnmente como el ensayo de Bradford 1, se utiliza ampliamente debido a su protocolo rápido y conveniente, así como de su sensibilidad relativa. Desafortunadamente, no existe un alto grado de curvatura en un amplio rango de concentraciones de proteína (Fig. 1). Por lo tanto, sólo una estrecha gama de concentraciones de proteína relativamente alto, 10.2 mg / ml de BSA, se utiliza para la curva de calibración, la cual se ajusta mejor a la regresión lineal (Fig. 1, color verde). Sin embargo, la no linealidad requiere la concentración de proteínas de las muestras desconocidas a caer dentro de la gama limitada de la curva de calibración con el fin de evitar un gran error, y también reduce la precisión en el alcance limitado. La no linealidad se presenta un problema serio, en particular, cuando las cantidades de microgramos de proteína no están disponibles, y con frecuencia requiere múltiples diluciones de las muestras desconocidas. Como se señaló en el documento original de Bradford, &quot;la fuente de la no linealidad es el reactivo en sí mismo, ya hay un solapamiento en el espectro de las dos formas de diferente color de la tintura.&quot; 1. De hecho, las tres formas del colorante azul brillante de Coomassie se encuentran en equilibrio ácido-base en el pH ácido normal de la prueba 2. Las formas de color rojo, azul y verde han absorbancia máxima a 470, 590 y 650 nm, respectivamente (Fig. 2). El azul es la forma que se une a la proteína, formando un complejo que absorbe la luz intensa a 594 nm 3, 4 (Fig. 2). Bradford también señaló que &quot;el valor de fondo para el reactivo disminuye constantemente a medida que más colorante se une a proteínas&quot; 1 (Fig. 3). Por lo tanto, tratamos de calcular la reducción del fondo de 590 nm como el aumento de cantidades de la proteína se agregan, midiendo el cambio de absorbancia a 450 nm, donde el complejo proteína-colorante no absorbe. Hemos encontrado que la disminución parcial de fondo, pero no totalmente, da cuenta de la no linealidad (Fig. 4). A continuación, la hipótesis de que la disminución de la concentración de colorante libre produce otra distorsión de la respuesta lineal, porque a medida que la proteína de fijación de colorantes está en equilibrio 5, la formación de complejos no sólo depende de la concentración de la proteína libre, sino también en el de la tintura libre (Fig. 5). Teniendo en cuenta tanto cuestiones relacionadas con la concentración variable del colorante libre, se desarrolló una ecuación matemática que describe una relación lineal entre la concentración de proteínas y la relación de medidas de absorbancia, 590 nm más de 450 nm (Fig. 6). Una descripción detallada del estudio teórico y experimental se puede encontrar en nuestra publicación en 1996 de Analytical Biochemistry 6. La ecuación matemática que fue probado experimentalmente y se encontró que el rendimiento de una curva de calibración lineal en la proteína de toda la gama de concentraciones (Fig. 7). Por otra parte, la ecuación también fue validado por una determinación independiente de la correcta depende del pH valor de la intersección del eje 6. Protocolo detallado para el ensayo de proteína mejorada Bradford, utilizando un lector de microplacas Absorbancia: <ol><li> Preparar un 0,1 mg / ml solución madre de la norma, la albúmina sérica bovina. Cualquier otra norma puede ser elegido, pero tenga en cuenta que la misma norma debe ser utilizado en todos los experimentos. </li><li> Diluir las muestras desconocidas en agua destilada. Objetivo de 50 a 50 mg / ml. Sin embargo, mayor o menor concentración de proteínas son aceptables, ya que no existe un límite claro para el rango lineal del ensayo. Sin embargo, la medición debe estar dentro del rango lineal del lector de absorbancia. </li><li> Diluir el reactivo de Bradford (Bio-Rad) de 2,5 veces en agua destilada. </li><li> Añadir 0 y 10 a 50 l de solución stock de BSA para tres pozos (la creación de un 0-5 mg curva de calibración BSA). Complementar con agua destilada para llegar a 100 l / pocillo. </li><li> En diferentes pozos, añadir 100 ml de la muestra problema por triplicado. Varias concentraciones de la muestra desconocida puede ser utilizado para aumentar la precisión. </li><li> Añadir 100 ml de los gases de reactivo de Bradford a todos los pocillos. El volumen total es de 200 l / pocillo. </li><li> Espere por lo menos 5 minutos, pero no más de 60 minutos para que el color. </li><li> El blanco debe ser de 200 l de agua destilada (y no el tinte de proteínas y cero). </li><li> Medir la absorbancia a 590 nm y 450 nm. </li><li> Preparar una curva de calibración dividiendo los valores de absorbancia neta a 590 nm y 450 nm. Tenga en cuenta que la proteína cero (tinte solamente) el valor debe ser incluido como un punto de datos (Fig. 8). </li><li> Calcular la concentración de la muestra problema sobre la base de la ecuación lineal de la curva de calibración (Fig. 9). </li></ol> Los resultados representativos: A diferencia de la absorbancia a una longitud de onda de 590 nm, la proporción de valores de absorción, 590 nm más de 450 nm, es lineal wconcentración de proteína i (Fig. 10). La concentración de proteína de la muestra desconocida puede ser simplemente calculó mediante la ecuación lineal de la curva de calibración (Fig. 10, la ecuación). Sin embargo, una mayor precisión se obtiene mediante la medición de varias diluciones de la muestra desconocida. Con este fin, preparar dos gráficas. La primera es una curva de calibración para el estándar de proteínas g en el eje X (Fig. 11, derecha). El segundo gráfico es que la muestra desconocida, con l de la muestra original sin diluir en el eje X (Fig. 11, izquierda). El único valor medio de contraste debe ser incluido en ambos gráficos (Fig. 11). La concentración de proteína de la muestra desconocida se obtiene dividiendo las laderas de la muestra desconocida y el estándar (Fig. 11, la ecuación). <img src="/files/ftp_upload/1918/1918fig2.jpg" alt="Figura 1" /> Figura 1. Gráfica convencional de calibración de Bradford. El rango lineal es representada por símbolos de color verde. <img src="/files/ftp_upload/1918/1918fig1.jpg" alt="Figura 2" /> Figura 2. Espectros del complejo proteína-colorante y el colorante solo. <img src="/files/ftp_upload/1918/1918fig3.jpg" alt="Figura 3" /> Figura 3. Proteína-colorante equilibrio. <img src="/files/ftp_upload/1918/1918fig4.jpg" alt="Figura 4" /> Figura 4. Reducción de los antecedentes correctos calculado disminuye parcialmente la no linealidad. <img src="/files/ftp_upload/1918/1918fig5.jpg" alt="Figura 5" /> Figura 5. Proteína-colorante equilibrio. <img src="/files/ftp_upload/1918/1918fig6.jpg" alt="Figura 6" /> Figura 6. Base matemática para la linealización de la proteína de ensayo Bradford. <img src="/files/ftp_upload/1918/1918fig7.jpg" alt="Figura 7" /> Figura 7. Linealización de la curva de calibración Bradford. <img src="/files/ftp_upload/1918/1918fig8.jpg" alt="Figura 8" /> Figura 8. Cálculo de los valores de absorbancia relación. <img src="/files/ftp_upload/1918/1918fig9.jpg" alt="Figura 9" /> Figura 9. Un gráfico lineal Bradford calibración. <img src="/files/ftp_upload/1918/1918fig10.jpg" alt="Figura 10" /> Figura 10. Linealización de la curva de calibración Bradford. <img src="/files/ftp_upload/1918/1918fig11.jpg" alt="Figura 11" /> Figura 11. Desconocido muestra la concentración de cálculo.

<ol>
	<li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Chial, H. J., Thompson, H. B., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spectral+study+of+the+charge+forms+of+Coomassie+blue+G.">A spectral study of the charge forms of Coomassie blue G.</a> Anal Biochem. 209, 258-266 (1993).</li><li>Chial, H. J., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+binding+of+Coomassie+brilliant+blue+to+proteins+at+low+and+neutral+pH.">A comparison of the binding of Coomassie brilliant blue to proteins at low and neutral pH.</a> Anal Biochem. 213, 362-369 (1993).</li><li>Compton, S. J., Jones, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+dye+response+and+interference+in+the+Bradford+protein+assay.">Mechanism of dye response and interference in the Bradford protein assay.</a> Anal Biochem. 151, 369-374 (1985).</li><li>Congdon, R. W., Muth, G. W., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+binding+interaction+of+Coomassie+blue+with+proteins.">The binding interaction of Coomassie blue with proteins.</a> Anal Biochem. 213, 407-413 (1993).</li><li>Zor, T., Selinger, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+Bradford+protein+assay+increases+its+sensitivity:+theoretical+and+experimental+studies.">Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies.</a> Anal Biochem. 236, 302-308 (1996).</li></ol>

El ensayo de proteínas Bradford es muy popular debido a su facilidad de realización y la sensibilidad relativa. La linealización sobre las concentraciones de proteína completa gama que se obtenga el protocolo que aquí se presenta simplifica aún más el ensayo, ya que las muestras desconocidas no tiene que estar dentro del rango de la curva de calibración. Es importante destacar que el protocolo mejorado aún ofrece las siguientes ventajas sobre el protocolo original de Bradford: <...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bradford reagent</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Amersco</td>
<td>0332</td>
<td>&nbsp;</td>
<tr>
<td>Multiplate absorbance reader</td>
<td>&nbsp;</td>
<td>BioTek</td>
<td>Synergy2</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

linearization of the bradford protein assay

Determinación de las cantidades de microgramos de proteína en el ensayo de Bradford azul brillante de Coomassie se lleva a cabo mediante la medición de la absorbancia a 590 nm. Este ensayo más común permite la cuantificación de proteínas rápida y sencilla en lisados ​​de células, las fracciones celulares, o muestras de proteínas recombinantes, con el propósito de la normalización de las mediciones bioquímicas. Sin embargo, una no-linealidad intrínseca compromete la sensibilidad y la precisión de este método. Se demuestra que bajo condiciones de ensayo estándar, la proporción de las mediciones de absorbancia a 590 nm y 450 nm es estrictamente lineal con la concentración de proteínas. Este sencillo procedimiento aumenta la precisión y mejora la sensibilidad de la prueba unas 10 veces, permitiendo la cuantificación de hasta 50 ng de albúmina sérica bovina. Además, la interferencia común introducido por los detergentes que se utilizan para crear los lisados ​​celulares se reduce considerablemente por el nuevo protocolo. Una ecuación lineal desarrollado sobre la base de la acción de masas y la ley de Beer se adapta perfectamente a los datos experimentales.

Watch this Scientific Journal Video about Linearization of the Bradford Protein Assay at JoVE.com

Linearization of the Bradford Protein Assay

La precisión y la sensibilidad de la determinación de proteínas mediante el ensayo de Bradford rápida y conveniente se ve comprometida por la no linealidad intrínseca. Se muestra un procedimiento de linealización simple que aumenta en gran medida la precisión, mejora la sensibilidad de la prueba unas 10 veces, y reduce significativamente la interferencia de los detergentes.

Linealización del ensayo de proteínas Bradford

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. ...

linearization-of-the-bradford-protein-assay

Research

JoVE Journal

Biology

97.9K vistas.  Tel Aviv University. La precisión y la sensibilidad de la determinación de proteínas mediante el ensayo de Bradford rápida y conveniente se ve comprometida por la no linealidad intrínseca. Se muestra un procedimiento de linealización simple que aumenta en gran medida la precisión, mejora la sensibilidad de la prueba unas 10 veces, y reduce significativamente la interferencia de los detergentes.

Video: Linearization of the Bradford Protein Assay

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Investigación

Biología

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

La detección de ubiquitinación de proteínas

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

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

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

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

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 ...

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

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

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

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolecular complementación de fluorescencia (BiFC) Ensayo de interacción proteína-proteína en las células de cebolla con la pistola de genes Helios

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Una afinidad líquido fase de captura de ensayo utilizando partículas magnéticas para estudiar la interacción proteína-proteína: el ejemplo de poliomyelits-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

Vinculación de riesgo de depredación, Estrés Fisiológico herbívoro y descomposición microbiana de los desechos de las plantas

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Ensayo de crecimiento basado en reportero para el Análisis Sistemático de la degradación de proteínas

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Cuantitativa inmunofluorescencia Ensayo para medir la variación en los niveles de proteína en centrosomas

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

La movilidad electroforética cambio de ensayo (EMSA) para el estudio de las interacciones RNA-proteína: El IRE / IRP Ejemplo

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...