1. S. cerevisiae Cultures and Protein Expression
Note: A&#946; variants exhibit different relative aggregation propensities due to a mutation in a single residue at position 19 (Phe19) of the A&#946;42 peptide (Figure 1A). These peptide variants are tagged with green fluorescent protein (GFP), which acts as an aggregation reporter (Figure 1)29.
<ol>
	<li>Transform plasmids encoding for 20 A&#946;42-GFP variants into yeast cells with a BY4741 parental background (MATa his3&#916;1 leu2&#916;0 met15&#916;0 ura3&#916;0)30. Each plasmid encodes for an A&#946;42 mutant fused to GFP by a GSSGSSG linker. The plasmids are pESC(-URA) and include the selectable marker URA329.
	<ol>
		<li>Prepare the synthetic complete medium without uracil (SC-URA) with the following components: 1.9 g/L yeast nitrogen base without uracil, 5 g/L ammonium sulfate, and 900 mL of ddH2O combined with 100 mL of 20% (w/v) glucose.</li>
		<li>Put one colony of the transformed yeast cells into 20 mL of the SC-URA medium containing 2% glucose and grow the cultures overnight at 30 &#176;C under agitation at 210 rpm.</li>
	</ol>
	</li>
	<li>The next day, grow new yeast cells cultures by inoculating 100 &#181;L of the overnight culture into 5 mL of fresh SC-URA medium.</li>
	<li>At an OD590 of 0.5, centrifuge the cultures at 3,000 x g for 4 min, discard the supernatant, and resuspend the cells in the same volume of fresh SC-URA medium containing 2% raffinose.</li>
	<li>Incubate at 30 &#176;C under agitation at 210 rpm for 30 min. Then, centrifuge the cells at 3,000 x g for 4 min and discard the supernatant. Resuspend the cells in fresh SC-URA medium containing 2% galactose to induce the recombinant protein expression.</li>
	<li>After 16 h of inducing protein expression in SC-URA medium containing 2% galactose, harvest 1 mL of the induced cells in sterile microcentrifuge tubes by centrifugation at 3,000 x g for 4 min. 
	NOTE: The most relevant differences occur after an induction period of 16 h. Induced yeast cells expressing A&#946;42-GFP variants can be visualized by fluorescence microscopy to determine the recombinant protein distribution inside the cells (Figure 2B).</li>
</ol>
2. Cell Staining
Note: Fresh non-induced cells are required as a negative control to establish a fluorescent threshold during an FC analysis.
<ol>
	<li>Take the 16 h-induced yeast cells and determine their optical density at 590 nm (OD590). Dilute them in a sterile phosphate buffered saline (PBS) to an OD590 of 0.1.
	<ol>
		<li>Prepare 1x phosphate buffered saline as follows: add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 to 800 mL of ddH2O. Fill the solution up to 1 L of ddH2O after adjusting the pH to 7.4 with HCl. Filter the buffer through a 0.2 &#181;m filter.</li>
	</ol>
	</li>
	<li>Transfer the expressing cell suspensions to appropriately labeled tubes (12 &#215; 75 mm round-bottom polystyrene) and protect them from light. Make sure they are ready to be loaded on the flow cytometer for analysis, together with non-induced and non-stained cells.</li>
	<li>Add the oxidative stress probe (e.g., CellROX Deep Red) to each sample at a final concentration of 5 &#181;M and incubate the cells at 30 &#176;C for 30 min in the dark.</li>
	<li>After incubation, wash the cells 3 times with 1x PBS and resuspend them in the same volume of buffer before their analysis by FC.</li>
</ol>
3. Flow Cytometry Analysis
Note: Use an FC setup with appropriate lasers and filters to detect GFP and oxidative stress probe fluorescence signal. A flow cytometer equipped with a 488 nm blue laser for the detection of GFP and a 635 nm red laser for the detection of oxidative stress probe fluorescence can be used. Acquisition of emission fluorescence of GFP and oxidative stress probe is performed with a 530/30 nm BP filter and a 660/20 BP filter, respectively (Table 1).
<ol>
	<li>Click on Open New Worksheet Panel and create the following acquisition dot plots.
	<ol>
		<li>Select the Scatter Plot Tool from the toolbar and create a plot with the variables Side Scatter-Area (SSC-A) on the y-axis vs. Forward Scatter-Area (FSC-A) in a linear scale on the x-axis.</li>
		<li>Click the Scatter Plot Tool from the toolbar and create a plot with the variables FL1-Area (FITC) on the x-axis vs. FL3-Area (APC) in a logarithmic scale on the y-axis.</li>
	</ol>
	</li>
	<li>Click the Instruments Settings icon. Click the Compensation tab and set all compensation levels. Click on the Acquisition tab and select a total number of 20,000 events to be recorded.</li>
	<li>Click the Flow Rate icon to switch the flow rate to Low.</li>
	<li>Click the Acquire tab to start running the non-induced, unstained cells and adjust the voltage in the Instrument Settings of the forward and side scatter until the population is distributed in the middle-left quadrant.
	<ol>
		<li>Click on the Polygon icon to set a region R1 around the cell population excluding cell debris, and use this gated population P1 = R1 for all fluorescent dot plots and histogram representations (Figure 3).</li>
	</ol>
	</li>
	<li>To adjust the PMT voltage of the fluorescence signal, run the unstained cells in the FL1-FL3 dot plot, tuning the gain in the Instrument Setting tab, until the cells are distributed in the lower left quadrant.</li>
	<li>Change the sample to the induced cells to measure GFP fluorescence. In the Instrument Setting tab, set the gain in the FSC-A vs. FL1 (FITC) when the population is distributed in the lower-right quadrant. Define the positive cell population with a gate (Gate P2).</li>
	<li>Change the sample to the non-induced stained cells to display oxidative stress by the fluorescence on an FSC-A vs. FL3 (APC) dot plot. Tune the gain until the cell population is distributed in the left-upper quadrant. Gate the positive cell population in P3.</li>
	<li>Make two histogram plots with the histogram icon to represent the cell fluorescence. To represent the fluorescence intensity, make sure FL1 (FITC) representing the GFP fluorescence is in the x-axis, and FL3 (APC) representing the fluorescence is in the y-axis, applying P2 and P3 populations on a logarithmic scale, respectively. 
	NOTE: Histogram overlays are a good way to illustrate differences between the fluorescence profiles of cells expressing different A&#946; variants.</li>
</ol>
4. Recombinant Protein Immunodetection
<ol>
	<li>To determine protein expression levels, harvest the yeast cultures induced for 16 h by centrifugation at 4,000 x g for 6 min, and store the cell pellets at -80 &#176;C.
	<ol>
		<li>Resuspend the collected cells expressing recombinant protein for 16 h in PBS. Prepare 200 &#181;L cell suspensions of each A&#946;42-GFP mutant to an OD590 of 20.</li>
	</ol>
	</li>
	<li>For total protein fraction analysis, harvest 100 &#181;L of each mutant by centrifugation at 14,000 x g for 20 min and resuspend the cell pellets in the same volume of yeast lysis buffer Y-PER (50 mM Tris-HCl pH 8.0, 1% DMSO, 200 mM NaCl, 1 mM EDTA containing 10 mg/mL SB3-14 (myristyl sulfobetaine) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF)).
	<ol>
		<li>Incubate the samples for 20 min at room temperature under mild agitation.</li>
	</ol>
	</li>
	<li>Determine the protein extract concentration by using the Bradford assay. Load up to 5 &#181;g of protein extract of each sample on a 15% acrylamide SDS-PAGE electrophoresis gel and blot onto a polyvinylidene difluoride (PVDF) membrane at 100 V for 60 min.
	<ol>
		<li>Prepare a Bradford reagent with 100 mg of Coomassie Brilliant Blue G-250 dissolved in 50 mL of 95% ethanol and add 100 mL of 85% (w/v) phosphoric acid. Then, when the dye is completely dissolved, dilute the mixture with ddH2O to 1 L and filter it through cellulose filter paper just before use. 
		NOTE: The Bradford reagent should be light brown.</li>
		<li>Prepare a standard of bovine serum albumin (BSA) ranging from 5 - 100 &#181;g of protein in 1,000 &#181;L of the Bradford reagent.</li>
		<li>Add 1 -&#160;10 &#181;L of protein extract to 1 mL of the Bradford reagent and incubate the mixture at room temperature for 5 min.</li>
		<li>Measure the absorbance of the standards and the protein extract samples at OD595.</li>
		<li>For the analysis, make a plot with the absorbance values obtained for the standards vs. &#181;g of protein. Based on the standard curve, determine the concentrations of the original samples from the amount of protein, considering the volume and dilution, if any.</li>
	</ol>
	</li>
	<li>Rinse the membrane with 1x Tris-buffered saline (TBS), 0.1% Tween 20 (TTBS), and block it using 5% (w/v) nonfat dry milk in 1x TTBS.
	<ol>
		<li>To prepare 1 L of 10x TBS, dissolve 24 g of Tris base and 88 g of NaCl in 900 mL of H2Odd and adjust the pH to 7.6. Add ddH2O to a final volume of 1 L. For a 1x solution, mix 1 part of 10x stock with 9 parts of ddH2O.</li>
	</ol>
	</li>
	<li>Incubate the membrane for 1 h with a primary &#946;-amyloid antibody 6E10 diluted 1:1,000 and for 1 h at room temperature with a secondary antibody goat anti-mouse IgG-HRP conjugate diluted 1:10,000.</li>
	<li>Develop membranes using 1 mL of Luminata fluorescent reagent and quantify bands by densitometric analysis as described in step 5.2.</li>
</ol>
5. Data Analysis
<ol>
	<li>To analyze the data obtained by FC, create a table displaying the mean fluorescent intensity (MFI) and median fluorescence with its corresponding standard error and/or the coefficient of variance (CV) for GFP fluorescence and oxidative stress levels.
	<ol>
		<li>Click the Inspector tab and the Statistics icon to customize the statistical data that are required for each channel. 
		NOTE: When comparing protein variants, convert fluorescence values data to a fold-over background or resolution metric (RD): 
		<img alt="Equation 1" src="/files/ftp_upload/57470/57470eq1.jpg" /> 
		In this metric, the mean sample is the mean fluorescence of problem samples, the mean control is the mean fluorescence of cells used as a control, and the SD is the standard deviation of the mean fluorescence. RD factor is a more accurate index than MFI value as it not only calculates the difference between samples but also accounts for the spreading of the data. This calculation is specifically relevant when comparing data coming from different experiments carried out over time. Once the RDs have been calculated, the use of a statistical test is advisable to confirm the significance of the observed differences between protein variants.</li>
	</ol>
	</li>
	<li>For protein immunodetection, quantify the recombinant protein levels by Western blot using ImageJ software.
	<ol>
		<li>Before the densitometry analysis, convert the original raw images of the developed membrane into JPEG file format and greyscale mode.</li>
		<li>Adjust the Set Measurements section of the Analyze menu to Mean Grey Value.</li>
		<li>Click on the Rectangle tool from ImageJ and define a region of interest (ROI) with a consistent size for the band densitometry analysis. Center each band on the membrane image inside the created rectangular frame and record the measurement one by one using Ctrl+M (the measurement command).</li>
		<li>Measure the local background adjacent to each band, applying the same ROI area. After measurement, subtract the corresponding local background from each individual band.</li>
	</ol>
	</li>
</ol>

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A wide range of diseases is linked to the accumulation of misfolded proteins into cellular deposits6,7,8,33. Many efforts have been made to unravel the molecular mechanisms that trigger the onset of these diseases using computational approaches, which do not take into account protein concentrations, or in vitro approaches, in which the protein concentration remains constant during the ...

Proteostasis is a fundamental determinant of cell fitness and aging processes. In cells, protein homeostasis is maintained by sophisticated protein quality control networks aimed to ensure the correct refolding of misfolded protein conformers by chaperones and/or their targeted proteolysis with several well-conserved mechanisms1,2,3,4,5. A large number of studies provide support to the link between the onset and progression of a broad range of human diseases and the failure of proteostasis, leading to protein misfolding and aggregation. For instance, the presence of protein deposits is considered a pathological hallmark of many neurodegenerative disorders, such as Alzheimer&#39;s, Parkinson&#39;s, and Huntington&#39;s diseases6,7,8, prionogenic diseases, and non-degenerative amyloidoses9. It has been suggested that early oligomeric and protofibrillar assemblies in the aggregation reaction are the main elicitors of cytotoxicity, establishing aberrant interactions with other proteins in the crowded cellular milieu10. In addition, protein inclusions (PI) can be transmitted between cells, propagating their toxic effect11,12. Therefore, it could be that the formation of PI might indeed constitute a detoxifying mechanism that restricts the presence of dangerous aggregated species to specific locations in the cell, where they can be processed or accumulated without major side effects13,14.
Standard in vitro biochemical approaches have provided important insights into the different species that populate aggregation reactions and their properties15,16. However, the conditions used in these assays are clearly different from those occurring within the cell and, therefore, question their physiological relevance. Because of the notable conservation of cellular pathways such as protein quality control, autophagy, or the regulation of the cellular redox state17,18 among eukaryotes19,20,21,22,23, the budding yeast Saccharomyces cerevisiae (S. cerevisiae) has emerged as a privileged simple cellular model to study the molecular determinants of protein aggregation and its associated cytotoxic impact in biologically relevant environments24,25,26.
Protein aggregation propensity is a feature inherently encoded in the primary sequence. Thus, the formation of amyloid-like structures can be predicted based on the identification and evaluation of the potency of aggregation-promoting regions in polypeptides27. However, despite the success of bioinformatic algorithms to predict the in vitro aggregation properties of protein sequences, they are still far from forecasting how these propensities translate into in vivo cytotoxic impact. Studies that address the link between the aggregated state of a given protein and its associated cellular damage in a systematic manner may help to circumvent this computational limitation. This connection is addressed in the present study, taking advantage of a large set of variants of the amyloid-&#946; peptide A&#946;42 differing only in a single residue, but displaying a continuous range of aggregation propensities in vivo28. In particular, an FC-based approach to identify the conformational species accounting for the oxidative damage elicited by aggregation-prone proteins in yeast cells is described. The methodology provides many advantages such as simplicity, high-throughput capability, and accurate quantitative measurement. This approach made it possible to confirm that PI play a protective role against oxidative stress.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Yeast cells BY4741&#160;</td>
			<td>ATCC</td>
			<td>201388</td>
			<td>Genotype: MATa his3&#916;1 leu2&#916;0 met15&#916;0 ura3&#916;0</td>
		</tr>
		<tr>
			<td>pESC(-Ura) plasmid&#160;</td>
			<td>Agilent Genomics</td>
			<td>217454</td>
			<td>Yeast expression plasmid with a Gal promotor. Selectable marker URA3</td>
		</tr>
		<tr>
			<td>Yeast Synthetic Drop-out Medium Supplements</td>
			<td>Sigma</td>
			<td>Y1501</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base Without Amino Acids</td>
			<td>Sigma</td>
			<td>Y0626</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Raffinose</td>
			<td>Sigma</td>
			<td>R7630</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma</td>
			<td>G0750</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP3991&#160;</td>
			<td>Solution 10X</td>
		</tr>
		<tr>
			<td>CellROX Deep Red Reagent&#160;</td>
			<td>Life Technologies</td>
			<td>C10422</td>
			<td>Free radical cell-permeant fluorescent sensor, non-fluorescent while in a reduced state, and exhibits bright fluorescence upon oxidation by reactive oxygen species (ROS), with absorption/emission maxima at 644/665 nm.&#160;</td>
		</tr>
		<tr>
			<td>Y-PER protein extraction reagent&#160;</td>
			<td>Thermo Scientific</td>
			<td>78990</td>
			<td>Liquid cell lysis buffer</td>
		</tr>
		<tr>
			<td>Acrylamide/Bis-acrylamide</td>
			<td>Sigma</td>
			<td>A6050</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Bradford dye reagent</td>
			<td>Bio-Rad&#160;</td>
			<td>5000205</td>
			<td>Dye reagent for one-step determination of protein concentration</td>
		</tr>
		<tr>
			<td>&#946;-amyloid antibody 6E10&#160;</td>
			<td>BioLegend</td>
			<td>803001</td>
			<td>Mouse IgG1. The epitope lies within amino acids 3-8 of beta amyloid (EFRHDS).</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HRP conjugate&#160;</td>
			<td>Bio-Rad</td>
			<td>1721011</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Immobilon-P, PVDF</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminata forte</td>
			<td>Merk</td>
			<td>WBLUF0100</td>
			<td>Premixed, ready to use chemiluminescent HRP detection reagent</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride solution (PMSF)</td>
			<td>Sigma</td>
			<td>93482</td>
			<td>Protease inhibitor. Dissolved at 0.1 M in ethanol</td>
		</tr>
		<tr>
			<td>FACSCanto flow cytometer&#160;</td>
			<td>BD Biosciences</td>
			<td>657338</td>
			<td>Equipped with a 488&#160;nm blue laser for the detection of GFP, and 635&#160;nm red laser / 530/30 nm BP filter and 660/20 BP filter</td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Electrophoresis Transfer cell</td>
			<td>Bio-Rad</td>
			<td>1703930</td>
			<td>Protein transference system</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Handcast Systems</td>
			<td>Bio-Rad</td>
			<td>1658000FC</td>
			<td>Electrophoresis system</td>
		</tr>
	</tbody>
</table>

evaluation of the impact of protein aggregation on cellular oxidative stress in yeast

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-&#946; peptide variants with their respective intrinsic aggregation propensities.

This protocol describes how to employ a collection of 20 variants of the A&#946;42 peptide where Phe19 has been mutated to all natural proteinogenic amino acids28. The theoretical aggregation propensities of these proteins can be analyzed using two different bioinformatic algorithms (AGGRESCAN and TANGO31,32). In both cases, this analysis renders a progressive gradation of aggregation tendencies, ascribing,...

Watch this Scientific Journal Video about Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast at JoVE.com

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-&#946; peptide.

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

evaluation-impact-protein-aggregation-on-cellular-oxidative-stress

Research

JoVE Journal

Biochemistry

7.2K Views.  Universitat Autònoma de Barcelona. Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-β peptide.

Video: Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...