Este artigo é dedicado à memória do falecido Dr. Zvi Selinger, que sediou a pesquisa original descreveu aqui.

Linearização da curva de calibração Protein Bradford: O ensaio de proteína Coomassie azul brilhante, vulgarmente conhecido como o ensaio de Bradford 1, é amplamente utilizado devido ao seu protocolo rápida e conveniente, bem como a sua sensibilidade relativa. Infelizmente, não há um grande grau de curvatura sobre uma ampla gama de concentrações de proteína (Fig. 1). Portanto, somente uma estreita faixa de concentrações de proteína relativamente alto, 2-10 mg / ml BSA, é usado para o gráfico de calibração, que então se encaixa melhor regressão linear (Fig. 1, verde). No entanto, a não-linearidade exige concentração de proteína das amostras desconhecidas para cair dentro da faixa limitada do gráfico de calibração, a fim de evitar um grande erro, e também reduz a precisão dentro da faixa limitada. A não-linearidade apresenta um sério problema em particular quando as quantias microgramas de proteínas não estão disponíveis, e que muitas vezes requer várias diluições das amostras desconhecidas. Como observado no papel Bradford original, &quot;a fonte da não-linearidade é o reagente em si uma vez que há uma sobreposição no espectro das duas formas de cores diferentes do corante.&quot; 1. Na verdade, três formas de o corante Coomassie azul brilhante estão em equilíbrio ácido-base no pH ácido usual do ensaio 2. Os formulários de vermelho, azul e verde têm absorbância maxima de 470, 590 e 650 nm, respectivamente (Fig. 2). O azul é a forma que se liga a proteínas, formando um complexo que absorve a luz intensa em 594 nm 3, 4 (Fig. 2). Bradford também observou que &quot;o valor de fundo para o reagente está continuamente diminuindo à medida que mais corante é ligada à proteína&quot; 1 (Fig. 3). Portanto, buscou-se calcular a redução do fundo nm 590 como quantidades de proteína crescente são adicionados, medindo a variação de absorvância a 450 nm, onde o complexo proteína-corante não absorve. Nós descobrimos que o fundo diminuindo parcialmente, mas não totalmente, as contas para a não-linearidade (Fig. 4). Em seguida, a hipótese de que a diminuição da concentração de corante livre produz outra distorção da resposta linear, porque como proteínas dye-binding está em equilíbrio 5, a formação do complexo não depende apenas da concentração da proteína livre, mas também na da tintura livre (Fig. 5). Tendo em conta as questões relacionadas com a concentração variável do corante livre, nós desenvolvemos uma equação matemática que descreve uma relação linear entre a concentração de proteína ea relação de medidas de absorbância, 590 nm mais de 450 nm (Fig. 6). Uma descrição detalhada do estudo teórico e experimental podem ser encontrados em nossa publicação de 1996, em Analytical Biochemistry 6. A equação matemática foi experimentalmente testado e está a produzir uma curva de calibração linear em toda a faixa concentrações de proteína (Fig. 7). Além disso, a equação foi validada também por uma determinação independente do valor do pH-dependente correta da interceptação do eixo Y 6. Protocolo detalhado para o ensaio Melhor Proteína Bradford, utilizando um leitor de Absorbância Microplaca: <ol><li> Prepare uma solução estoque 0,1 mg / ml do padrão, albumina de soro bovino. Qualquer outra norma pode ser escolhido, mas note que o mesmo padrão deve ser utilizado em todos os experimentos. </li><li> Diluir as amostras desconhecidas em água deionizada. Objetivo de 50-50 mcg / ml. No entanto, as concentrações de proteína maior ou menor são aceitáveis, já que não há limite aparente para o intervalo linear do ensaio. No entanto, a medição deve estar dentro da faixa linear do leitor absorbância. </li><li> Diluir o reagente de Bradford (Bio-Rad) de 2,5 vezes em água deionizada. </li><li> Adicionar 0 e 10-50 mL de solução estoque de BSA poços triplicado (criação de uma curva de calibração 0-5 mg BSA). Complementar com água deionizada para alcançar 100 l / poço. </li><li> Em poços diferentes, adicione 100 ml da amostra desconhecida em triplicatas. Várias concentrações da amostra desconhecida pode ser usado para aumentar a precisão. </li><li> Adicionar 100 ul do reagente de Bradford diluído para todos os poços. Volume total é de 200 mL / poço. </li><li> Espere pelo menos 5 min, mas não mais de 60 min para o desenvolvimento de cor. </li><li> O branco deve ser de 200 mL de água deionizada (e não o corante de proteínas de zero também). </li><li> Medir a absorvância a 590 nm e 450 nm. </li><li> Prepare uma curva de calibração, dividindo os valores de absorbância líquida a 590 nm e 450 nm. Note-se que a proteína zero (corante apenas) o valor deve ser incluído como um ponto de dados (Fig. 8). </li><li> Calcular a concentração da amostra desconhecida com base na equação linear da curva de calibração (Fig. 9). </li></ol> Resultados representativos: Ao contrário da absorvância a um único comprimento de onda de 590 nm, a proporção de valores de absorbância, 590 nm mais de 450 nm, é linear wconcentração de proteína i (Fig. 10). A concentração de proteína da amostra desconhecida pode ser simplesmente calculado usando a equação linear da curva de calibração (Fig. 10, a equação). No entanto, uma maior precisão é obtida através da medição várias diluições de amostras desconhecidas. Para este efeito, prepare dois gráficos. O primeiro é um gráfico de calibração para o padrão com proteína mg no eixo X (Fig. 11, direita). O segundo gráfico é para a amostra desconhecida, com mL da amostra original não diluído no eixo X (Fig. 11, à esquerda). O valor da tintura só devem ser incluídos em ambos os gráficos (Fig. 11). A concentração de proteína da amostra desconhecida é derivado dividindo as pistas da amostra desconhecida e do padrão (Fig. 11, a equação). <img src="/files/ftp_upload/1918/1918fig2.jpg" alt="Figura 1" /> Figura 1. Gráfico de calibração convencional Bradford. A faixa linear é representado por símbolos verdes. <img src="/files/ftp_upload/1918/1918fig1.jpg" alt="Figura 2" /> Figura 2. Spectra do complexo proteína-corante e do corante sozinho. <img src="/files/ftp_upload/1918/1918fig3.jpg" alt="Figura 3" /> Figura 3. Protein equilíbrio dye. <img src="/files/ftp_upload/1918/1918fig4.jpg" alt="Figura 4" /> Figura 4. Redução do fundo correta calculada diminui parcialmente não-linearidade. <img src="/files/ftp_upload/1918/1918fig5.jpg" alt="Figura 5" /> Figura 5. Protein dye-equilíbrio. <img src="/files/ftp_upload/1918/1918fig6.jpg" alt="Figura 6" /> Figura 6. Base matemática para a linearização do ensaio de proteína Bradford. <img src="/files/ftp_upload/1918/1918fig7.jpg" alt="Figura 7" /> Figura 7. Linearização da curva de calibração Bradford. <img src="/files/ftp_upload/1918/1918fig8.jpg" alt="Figura 8" /> Figura 8. Cálculo dos valores de absorbância relação. <img src="/files/ftp_upload/1918/1918fig9.jpg" alt="Figura 9" /> Figura 9. A curva de calibração linear Bradford. <img src="/files/ftp_upload/1918/1918fig10.jpg" alt="Figura 10" /> Figura 10. Linearização da curva de calibração Bradford. <img src="/files/ftp_upload/1918/1918fig11.jpg" alt="Figura 11" /> Figura 11. Cálculo da amostra concentração Desconhecido.

<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>

O ensaio de proteína Bradford é popular devido à sua facilidade de desempenho e de sensibilidade relativa. A linearização sobre as concentrações de proteína toda a gama obtidos pelo protocolo aqui apresentado simplifica ainda mais o ensaio, como amostras desconhecidas não precisa estar dentro do intervalo da curva de calibração. Importante, o protocolo prevê ainda melhorou as seguintes vantagens sobre o original Bradford protocolo: <ol><li> Maior precisão. </li><li> A sensib...

<!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

Determinação das quantidades microgramas de proteína no ensaio Bradford Coomassie azul brilhante é realizado pela medição da absorbância a 590 nm. Este ensaio mais comum permite a quantificação de proteínas rápidas e simples em lisados ​​celulares, frações celulares, ou amostras de proteínas recombinantes, com a finalidade de normalização das dosagens bioquímicas. No entanto, uma não-linearidade intrínseca compromete a sensibilidade e precisão deste método. É mostrado que, sob condições de ensaio padrão, a relação entre as medidas de absorbância a 590 nm e 450 nm é estritamente linear com a concentração de proteína. Este simples procedimento aumenta a precisão e melhora a sensibilidade do ensaio cerca de 10 vezes quantificação, permitindo até 50 ng de albumina bovina. Além disso, a interferência comumente introduzidas por detergentes que são utilizados para criar a lisados ​​celulares é muito reduzida pelo novo protocolo. Uma equação linear desenvolvido com base em ação de massa e lei de Beer se encaixa perfeitamente os dados experimentais.

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

Linearization of the Bradford Protein Assay

A precisão e sensibilidade da determinação de proteína pelo ensaio Bradford rápida e conveniente é comprometida pela não-linearidade intrínseca. Nós mostramos um procedimento de linearização simples que aumenta a precisão, melhora a sensibilidade do ensaio cerca de 10 vezes, e reduz significativamente a interferência por detergentes.

Linearização do Ensaio de Proteína 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

98.2K visualizações.  Tel Aviv University. A precisão e sensibilidade da determinação de proteína pelo ensaio Bradford rápida e conveniente é comprometida pela não-linearidade intrínseca. Nós mostramos um procedimento de linearização simples que aumenta a precisão, melhora a sensibilidade do ensaio cerca de 10 vezes, e reduz significativamente a interferência por detergentes.

Vídeo: 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 Ensaio Co Sedimentação; para a Análise da proteína de ligação a 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 ...

Biologia

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.

Detecção de Ubiquitinação Protein

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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery 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 de fluorescência de Complementação Assay (BiFC) para Interação Proteína-Proteína em células de cebola Utilizando o Gene Gun 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.

A fase líquida ensaio de captura de afinidade usando pérolas magnéticas para estudar interação proteína-proteína: O Exemplo Poliovírus Nanobody-

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.

Ligando o risco de predação, Stress herbívoro fisiológica e decomposição da maca Usina

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.

Baseada Reporter ensaio de crescimento para análise sistemática de degradação 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.

Quantitative Reação de Imunofluorescência Indireta para medir a variação nos níveis de proteína no centrossomas

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.

Electrophoretic Mobility Turno Assay (EMSA) para o estudo das interações RNA em proteínas: O IRE / IRP Exemplo

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

Methods Article

Linearização do Ensaio de Proteína Bradford