We thank Dr. Yuval Reiss and Dr. William Breuer for critically reviewing the manuscript and Omri Alfassy for helping in the development of the Ura3-GFP screen assay. We also thank Dr. M. Hochstrasser and Dr. R. Kulka for plasmids and strains. This work was funded by the Israeli Academy of Sciences (grant 786/08) and by the United States-Israel Binational Scientific foundation (grant 2011253).

1. Cell Culture
<ol>
	<li>Transform the appropriate auxotrophic yeast cells, such as Try467 (Table 2), with a plasmid containing (a) a URA3-degron fusion and (b) an additional metabolic marker for plasmid selection and maintenance. 
	NOTE: An example of a suitable plasmid is YDpK-MET25p-Deg1-FLAG-Vma12-Ura3 (LYS2) (Deg1-UV). This is a yeast integrative plasmid containing a fusion protein comprising of Deg1 &#8212; a degron derived from the yeast transcription factor Mat217, FLAG epitope &#8212; for detection by immunoblotting, Vma12 &#8212; a stable ER protein, and Ura315,18 (Figure 3A). (See Table 3 for plasmids used in this study and for additional examples of suitable plasmids.)</li>
	<li>For plasmid selection and maintenance, keep cells in agar plates under a suitable Synthetic Defined (SD) medium lacking amino acid selection markers19 (For example, Deg1-UV is selected and maintained under SD-LYS selective media).</li>
	<li>Grow cells O/N at 30 &#176;C in a suitable selective SD medium for plasmid maintenance until a stationary phase is reached. Grow the cells in test tubes or in a 96-well plate, depending on the number of samples to be examined (section 2).</li>
</ol>
2. Cell Preparation for Analysis
<ol>
	<li>When a small number of samples (N&#8804;15) are to be assayed, dilute the cells and replace the medium as follows:
	<ol>
		<li>Dilute 50 &#181;l of cells from an O/N culture with 150 &#181;l of SD medium per well in a 96-well plate (clear bottom). Note, the first sample is designated as blank contains medium only.</li>
		<li>Determine the OD600 values of the samples in the 96-well plate using a microplate reader.</li>
		<li>Calculate the actual OD600 in each well by multiplying the OD600 reading by the dilution factor (x4), from which the value of the blank reading is subtracted. Normalize the resulting values by dividing by 0.55 to obtain a calculated path-length of 1 cm according to the Beer-Lambert law. Note, this value is constant when measuring the OD600 of 200 &#181;l of cells in a standard 96-well plate.</li>
		<li>Calculate the exact volume needed for obtaining yeast cells at amount equivalent to OD600 of 0.25 and transfer it to an Eppendorf tube.</li>
		<li>Spin down the cells at high speed (12,000 x g) for 1 min.</li>
		<li>Remove the supernatant and resuspend the cells in 1 ml of the appropriate selective medium (see section 3.1) to obtain OD600 of 0.25.</li>
		<li>Transfer 200 &#181;l of the diluted strains to a new well in a 96-well plate.</li>
	</ol>
	</li>
	<li>When a large number of samples (N&#62;15) are to be tested, dilute the cells and replace the medium without prior OD600 measurement, as follows:
	<ol>
		<li>Transfer 10 &#956;l of stationary cells grown O/N in a 96-well plate into a new 96-well plate containing 190 &#956;l (dilution 1:20) of the appropriate selective media (see section 3.1).</li>
	</ol>
	</li>
</ol>
3. Growth Kinetics in Liquid Culture Under Selective Conditions (GiLS)
<ol>
	<li>Use the following media for growth measurements using the Ura3-degron reporter:
	<ol>
		<li>For positive controls of yeast growth under non-restrictive conditions, use plasmid selective SD media (see section 1.2). If no plasmid selection is required, for example, when using an integrative plasmid such as Deg1-UV, SD medium containing all necessary amino acids (SD complete) can be used.</li>
		<li>For experimental samples measuring growth under restrictive conditions, use SD-URA medium.</li>
	</ol>
	</li>
	<li>Set a multimode microplate reader to: an incubation temperature of 30 &#176;C, OD600 measurement intervals of 15 min, and an orbital and linear shaking cycles of 1 min every seven min. 
	NOTE: This dual-mode shaking was optimized to obtain homogenous distribution of cells; however, other shaking modes or even no shaking may work as well.</li>
	<li>Incubate the cells at 30 &#176;C in a microplate reader O/N or until cells have reached the stationary phase (12-24 hr).</li>
	<li>Export the raw data to a spreadsheet file.</li>
	<li>Determine yeast growth kinetics using MDTcalc, a customized software program that calculates the minimal time (in hr) for cells to double their population size (Minimal Doubling Time (MDT); for details see section 4).</li>
</ol>
4. Calculation of Minimal Doubling Time (MDT): Principles of the MDTcalc Algorithm

NOTE: Principles of the MDTcalc algorithm, using a representative sample of Try467 yeast cells (Table 2) grown in SD complete media, are illustrated in Table 1 and Figure 1, showing a data spreadsheet and growth plots, respectively.

<ol>
	<li>Subtract OD600 blank values, obtained in the respective well, from each of the readings obtained in the sample well. Divide the resulting differences by 0.55 (for 96-well plates) to correct for the light path length. Plot the final values (Table 1, Trans.) against time to produce the actual yeast growth curve (OD600/hr) (Figure 1A).</li>
	<li>Calculate log2 (OD600) for each of the transformed values to convert the logarithmic growth phase into a linear curve (Figure 1B).</li>
	<li>Calculate the slope of a 10 time-points interval (N=10) starting at time 0 and moving one time-point at a time until N&#60;10 (Table 1, Slope; Figure 1C).</li>
	<li>Calculate the inverse value of each slope to obtain the time required for the cell population to double (Table 1, 1/Slope; Figure 1C). Calculate the minimal 1/Slope value to determine the MDT (Table 1, black rectangle; Figure 1C). Extract the calculated MDT for cells in the sample (Table 1, 1/Slope, black rectangle) from the selected time interval (Table 1, outlined by red rectangle, Figures 1B, 1D). 
	NOTE: The rate of cell growth on SD-URA medium is inversely proportional to the MDT.</li>
</ol>
5. Using MDTcalc
<ol>
	<li>Copy the supplemented program files into a designated folder in the computer.</li>
	<li>Make sure the spreadsheet file containing the growth data is saved and closed. Open the MDTcalc application program to see the start screen appears.
	<ol>
		<li>Insert the required information, as demonstrated in Figure 2:
		<ol>
			<li>Under &#39;File path&#39;, click on the rectangle on the right to locate the spreadsheet file.</li>
			<li>Under &#39;Sheet name&#39;, write the spreadsheet name where the raw data is located.</li>
			<li>Under &#39;Starting position&#39;, insert the spreadsheet coordinate of time 0 of the first sample.</li>
			<li>Under &#39;Time column&#39;, insert the letter defining the location of the time point column (time should be provided in seconds).</li>
			<li>Under &#39;Blank column&#39;, insert the letter defining the location of the blank column (usually but not necessarily in well A1 of the 96-well plate).</li>
			<li>Under &#39;OD value&#39;, choose the minimal transformed OD600 value required for the initiation of MDT calculation (usually 0.15-0.25). Setting this value prevents calculations of MDT for cultures in lag phase due to excessive dilution so that MDT is calculated only for samples that reach the defined OD600 value or higher.</li>
		</ol>
		</li>
		<li>Once the last value has been entered, press the &#39;calculate&#39; button at the bottom of the screen. Ensure that the progress bar on the right gradually changes to green. Do not open the spreadsheet file during the calculation process.</li>
		<li>Export the two sets of data that appear automatically on the screen at the completion of the MDT calculation into a spreadsheet. Ensure that the data includes: (a) the MDT value for each well that passes the minimal OD value test (as set in section 5.2.1.6) and (b) the initial time point of the interval from which the MDT was calculated (Start time).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here we describe an assay based on cell growth for determining relative protein degradation rates, termed &#39;Growth kinetics in Liquid culture under Selective conditions&#39; (GiLS). The GiLS assay has several advantages: It is simple to set up, data acquisition and analysis is straightforward and it is extremely modular. Consequently, GiLS can be applied simultaneously to multiple samples in a user friendly multi-well plate format that can be adapted to automation for high throughput applications. Most importantly, Gi...

El sistema de degradación ubiquitina-proteasoma es una máquina reguladora importante, que ha sido implicado en el mantenimiento de la homeostasis de la proteína en todos los eucariotas. El UPS conjuga inicialmente múltiples moléculas de ubiquitina a una proteína diana después de lo cual la proteína poli-ubiquitina-etiquetado es degradada por el proteasoma 26S. En la mayoría de los casos, el paso limitante de la degradación mediada por ubiquitina es ubiquitylation sustrato, mediada por la conjugación de enzimas E2 y E3 ligando enzimas (E3 ligasas) 1. En consecuencia, la estabilidad intracelular de proteínas específicas refleja su susceptibilidad a la ubiquitina-conjugación y la actividad de sus enzimas ubiquitylation afines. Ligasas E3 son los principales componentes de reconocimiento de sustrato de la UPS. Como tal, estas enzimas reconocen degrons dentro de sus sustratos que están ausentes o no expuestos en sus contrapartes estables 2. Por ejemplo, muchos reguladores del ciclo celular mUST ser sintetizado y degradado de manera temporal específica, a fin de mantener la progresión del ciclo celular en orden. La degradación de estas proteínas a menudo se controla por fosforilación, mediada por quinasas reguladas de señalización celular 3,4. Por otro lado, las proteínas plegadas de forma aberrante, se reconocen a través de degrons crípticos. Se trata de regiones que normalmente están ocultos en la estructura nativa y están expuestos a la estructura de la perturbación. Tales degrons incluyen dominios hidrofóbicos 5-7 y segmentos intrínsecamente desordenados 8. Desde el descubrimiento seminal del sistema-ubiquitina para la degradación de proteínas y la caracterización de sus fundamentos en lisados ​​de reticulocitos 9, la genética de la levadura fue instrumental en el descubrimiento de muchos de los componentes del sistema de ubiquitina 10. El éxito de la levadura como organismo modelo para el análisis sistemático de la degradación de proteínas por el UPS se debe principalmente al hecho de que el SAI es altoLy conserva en todos los eucariotas 4, junto con su amenability como un sistema experimental. De hecho, los sistemas basados ​​en levaduras se emplean comúnmente para descifrar los mecanismos de acción de la maquinaria de ubiquitinación. El estudio de la degradación de proteínas por medios bioquímicos por lo general requiere la preparación de extractos celulares. Mientras que las proteínas de células animales pueden ser extraídos en condiciones relativamente suaves que conservan las interacciones proteína y la función, la presencia de una pared celular de levadura robusto en condiciones de interrupción 11 requiere considerablemente más severas que pueden afectar a la recuperación de proteínas. De hecho, diferentes procedimientos para la interrupción de la célula de levadura varían considerablemente en su capacidad para recuperar las proteínas intactas en cantidades que representan correctamente su abundancia relativa celular. Además inexactitud inherente a los diferentes métodos utilizados para la determinación de las tasas de degradación de proteínas específicas: experimentos metabólicos basado etiquetado &#39;pulso-caza&#39; seguido de immunoprecipitation, para aislar proteínas específicas 12, a menudo no es estrictamente cuantitativo. Por lo tanto, cuando la degradación de proteínas se compara con este método, la precaución adicional debe tener precaución en la interpretación de los resultados. Para evitar este inconveniente, una alternativa cicloheximida (CHX) ensayo de persecución se puede emplear 12. En este ensayo, se añade el inhibidor de la traducción a cultivos de células y los cambios temporales en los niveles de estado estable de proteínas son monitoreados posteriormente. Sin embargo, el uso de CHX se limita a las proteínas con vidas medias relativamente cortas (&lt;90 min), como la inhibición a largo plazo de la síntesis de proteínas es citotóxico. En particular, tanto de los ensayos mencionados anteriormente requieren el uso de anticuerpos específicos para la proteína, que no siempre están disponibles. Para superar estas limitaciones técnicas, los investigadores han desarrollado varios enfoques que no requieren la extracción de células y la manipulación directa de proteínas. Un enfoque se basa en el establecimiento de sí auxotróficocepas del st, obtenidos mediante la supresión de los genes que codifican las enzimas metabólicas esenciales. Tales genes incluyen HIS3, LEU2, LYS2 y TRP1, que codifica para las enzimas necesarias para la biosíntesis de aminoácidos, así como URA3 que codifica OMP descarboxilasa (Ura3), una enzima esencial de la biosíntesis de pirimidina ribonucleótido. Ura3 ha sido ampliamente utilizado en estudios de degradación de proteínas. En estos ensayos, la expresión constitutiva de Ura3 rescata crecimiento de las células ura3 en medio sin uracilo deficiente 13. En consecuencia, desestabilizando Ura3 a través de la fusión de un degron puede disminuir el crecimiento de células en medio mínimo carente de uracilo. Este método se ha utilizado en varios estudios de degradación de proteínas, incluyendo la identificación de determinantes de la degradación de 5, ligasas E3 14 y ubiquitylation auxiliar factores de 15 y el descubrimiento de nuevos UPS degrons 16. Todos estos métodos empleados crecimiento de las células en placas de agar como lectura del ensayo. Sin embargo, tque el criterio de crecimiento (crecimiento positivo / negativo), mientras robusta y eficiente, es sobre todo cualitativo y no proporciona información cuantitativa que es importante para evaluar la potencia de un degron o la contribución relativa de los diversos factores de degradación auxiliares. Por ello, hemos desarrollado y utilizado los vectores de levadura y los métodos de detección que permite un análisis sistemático y cuantitativo de la degradación de proteínas por el URA3 -degron sistema de fusión. El protocolo se basa en un ensayo fácil de manejar que mide la cinética de crecimiento en cultivo líquido bajo condiciones selectivas (GILS) y en la generación de curvas de crecimiento estándar. Cinética de crecimiento de la levadura se caracterizan por tres fases principales - el desfase, la exponencial (de registro) y la fase estacionaria. Cálculo de la cinética de replicación de levadura durante la fase de registro en condiciones selectivas, que se determina por los niveles de expresión de la Ura3-degron, proporciona una medición cuantitativa o imparcialf degradación de proteínas. Este método se puede aplicar a la medición y la comparación de las tasas de degradación de múltiples sustratos UPS simultáneamente en múltiples cepas y bajo diversas condiciones.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Name of Material/ Equipment</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:339px;">Comments/Description</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:184px;">Difco yeast nitrogen base w/o amino acids and ammonium sulfate</td>
			<td>BD Biosciences</td>
			<td>233520</td>
			<td rowspan="6" style="width:339px;">For yeast growth on SD minimal media.&#160; Amino acids to be supplied are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium sulfate</td>
			<td>Sigma - Aldrich</td>
			<td>A4418</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma - Aldrich</td>
			<td>16325</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenine</td>
			<td>Sigma - Aldrich</td>
			<td>A8626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Uracil</td>
			<td>Sigma - Aldrich</td>
			<td>U0750</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amino acids</td>
			<td>Highest purity available&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">96 well plates</td>
			<td style="width:149px;">Nunc</td>
			<td style="width:119px;">167008</td>
			<td>Any other compatible brand can be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Cyclohexamide&#160;</td>
			<td style="width:149px;">Sigma - Aldrich</td>
			<td style="width:119px;">C7698</td>
			<td>Working conc. 0.5 mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:184px;">Infinite 200 PRO series</td>
			<td style="width:149px;">Tecan</td>
			<td style="width:119px;"></td>
			<td style="width:339px;">For yeast incubation and OD 600 measurements. Any other compatible temp-controled reader can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">MDTcalc</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td>Experimental software</td>
		</tr>
	</tbody>
</table>

reporter-based growth assay for systematic analysis of protein degradation

La degradación de proteínas por el sistema ubiquitina-proteasoma (UPS) es un mecanismo regulador importante para la homeostasis de la proteína en todos los eucariotas. El enfoque estándar para la determinación de la degradación intracelular de proteínas se basa en ensayos bioquímicos para el seguimiento de la cinética de disminución de la proteína. Tales métodos son a menudo laborioso y consume mucho tiempo y por lo tanto no susceptible de experimentos dirigidos a evaluar múltiples sustratos y condiciones de degradación. Como alternativa, los ensayos basados ​​en el crecimiento de células se han desarrollado, que son, en su formato convencional, ensayos de punto final que no puede determinar cuantitativamente los cambios relativos en los niveles de proteína. Aquí se describe un método que determina fielmente los cambios en las tasas de degradación de proteínas mediante el acoplamiento a la cinética de crecimiento celular de la levadura. El método se basa en un sistema de selección establecido donde uracilo auxotrofía de URA3 -deleted células de levadura es rescatado por una proteína reportera expresada exógenamente, Compuesto de una fusión entre el gen URA3 esencial y determinante de degradación (degron). La proteína indicadora está diseñado de manera que su tasa de síntesis es constante mientras que su velocidad de degradación se determina por la degron. Como el crecimiento de células en medio sin uracilo deficiente es proporcional a los niveles relativos de Ura3, la cinética de crecimiento dependen enteramente de la degradación de la proteína reportera. Este método mide con precisión los cambios en la cinética de degradación de proteínas intracelulares. Se aplicó a: (a) Evaluación de la contribución relativa de los factores de ubiquitina-conjugación conocidos a la proteolisis (b) la conjugación de la enzima E2 estructura-función analiza (c) Identificación y caracterización de nuevos degrons. La aplicación del sistema basado en URA3 degron- trasciende el campo de la degradación de proteínas, ya que también se puede adaptar a los cambios de monitorización de los niveles de proteína asociadas con las funciones de otras vías celulares.

Investigating the role of enzymes of the Doa10 pathway in the degradation of a reporter substrate 
To test the validity of the GiLS method it was compared to a traditional degradation assay. This experiment assesses the relative contribution of components of the ER-membrane localized Doa10 E3-ligase complex20,21 to the degradation of the protein quality control reporter substrate Deg1-VU (Figure 3A). The Deg1-VU plasmid was integra...

Watch this Scientific Journal Video about Reporter-based Growth Assay for Systematic Analysis of Protein Degradation at JoVE.com

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

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 ...

reporter-based-growth-assay-for-systematic-analysis-protein

Research

JoVE Journal

Biology

10.6K vistas.  The Hebrew University of Jerusalem. 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.

Video: Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análisis de Crecimiento Humano Colonia células madre embrionarias

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Determinación basada en el crecimiento y en la confirmación bioquímica de Requisitos Genéticos para la degradación de proteínas en<em&gt; Saccharomyces cerevisiae</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Análisis cicloheximida persecución de degradación de proteínas en<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Los ensayos para la degradación de proteínas en células de mal plegadas

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

Un Sistema de Cultivo Hidropónico para el Análisis Simultáneo y Sistemático de Interacciones Moleculares de Plantas / Microbios y Señalización

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Análisis precisos, alto rendimiento de crecimiento bacteriano

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...