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EoE Sub Articles

1. Proteasome Activity Assay
NOTE: Proteasome activity can be measured in homogenized brain tissue using a slightly modified version of the 20S Proteasome Activity Kit. This assay does not directly measure the activity of complete 26S proteasome complexes. Rather, it measures the activity of the 20S core, meaning it can only serve as a proxy to understand the activity of the core itself as opposed to the entire 26S proteasome complex. The success of this assay declines with repeated freeze-thaw cycles and/or increasing levels of detergents, particularly ionic, and requires the use of a plate reader with a 360/460 (excitation/emission) filter set and heating capabilities up to 37 &#176;C.
<ol>
	<li>Plate reader settings: Pre-warm to 37 &#176;C and hold through the run.&#8203;
	<ol>
		<li>Set excitation to 360 and emission to 460. If the 96 well plate used is clear, set the optics position to Bottom. If a dark/black 96 well plate is used, set optics position to Top.</li>
		<li>Set older plate reader models to Auto-Gain under the fluorescent read options; newer models are preset for this. Program a kinetic run with a time of 2 h, scanning (reading) every 30 min.</li>
	</ol>
	</li>
	<li>Reconstitute the 10x assay buffer provided in the kit with 13.5 mL of ultrapure water. Add 14 &#181;L of 100 mM ATP to the now 1x buffer; this significantly enhances proteasome activity in the samples and improves assay reliability. The final 20S Assay buffer can be stored on ice or at 4 &#176;C until needed and is stable for several months.</li>
	<li>Reconstitute the AMC standard provided in kit with 100 &#181;L of DMSO. Perform this step in the dark or under low light conditions, as the standard is light sensitive.</li>
	<li>Create a standard curve of AMC using the reconstituted standard, in the dark or under low light conditions.
	<ol>
		<li>In separate 0.5 mL microcentrifuge tubes, add 16, 8, 6.4, 3.2, 1.6, 0.8, 0.4 and 0 &#181;L of the AMC standard, which corresponds to 20, 10, 8, 4, 2, 1, 0.5 and 0 &#181;M AMC concentration.</li>
		<li>To these tubes, in the same order, add 84, 92, 93.6, 96.8, 98.4, 99.2, 99.6 and 100 &#181;L of 20S Assay buffer. This creates a series of high to low AMC concentrations, which will be used for plate reader calibration and analysis of proteasome activity in the homogenized samples.</li>
		<li>Store all diluted standards on ice in the dark until needed.</li>
	</ol>
	</li>
	<li>Reconstitute the proteasome substrate (Suc-LLVY-AMC) provided in kit with 65 &#181;L of DMSO. Perform this step in the dark or under low light conditions, as the substrate is light sensitive. Create a 1:20 dilution of the proteasome substrate in a new 1.5 mL microcentrifuge tube using 20S Assay buffer. Store the diluted substrate on ice in the dark until needed. 
	NOTE: For example, if the plate will have 10 samples and 1 blank, you will need enough diluted substrate for 22 wells (with duplicates) at 10 &#181;L per well. This equates to 220 &#181;L need + 30 &#181;L for pipetting errors, requiring 12.5 &#181;L of substrate and 237.5 &#181;L of 20S Assay buffer.</li>
	<li>Thaw desired samples (if frozen) and add a normalized amount to a 96 well plate. Run each sample in duplicates. The amount of sample needed varies based on tissue preparation. Generally, 10-20 &#181;g is sufficient for any subcellular fraction.</li>
	<li>Bring the sample well volume to 80 &#181;L with ultrapure water. The amount added depends on the volume of sample added. For example, if sample 1 was 4.5 &#181;L of protein and sample 2 was 8.7 &#181;L, then the amount of water needed will be 75.5 &#181;L and 71.3 &#181;L, respectively. In two separate wells, add 80 &#181;L of water alone; these will be the Assay Blanks. 
	NOTE: In order to limit changes in protein volume due to pipetting errors, protein concentrations can be normalized to the least concentrated sample. This will allow use of the same sample volume in all conditions.</li>
	<li>Add 10 &#181;L of 20S Assay buffer to each well, including Assay Blanks. A repeater/automated pipette is recommended here to ensure consistent assay volume across wells.</li>
	<li>Optional: At this stage, introduce in vitro manipulations if desired; this will require that each sample has an additional 2 wells per treatment, including the vehicle. If so, add 5-10 &#181;L of drug/compound of interest to 2 of the sample wells and an equivalent volume of control/vehicle to another 2 wells. Place the plate onto the pre-warmed plate reader or into a 37 &#176;C incubator for 30 min.</li>
	<li>Turn off the lights or enter a dark room. Add all 100 &#181;L of diluted AMC standards to a new well; each standard will have a single well.</li>
	<li>In the dark, add 10 &#181;L of diluted proteasome substrate to wells containing sample and Assay Blanks, but not to the AMC standard. A repeater/automated pipette is recommended here to ensure consistent assay volume across wells.</li>
	<li>Place the plate into the plate reader and start the kinetic run. 
	NOTE: The plate does not need to be under constant agitation during the kinetic run, however, the user can choose to if desired</li>
	<li>At end of the kinetic run, export raw 360/460 fluorescent values to Microsoft Excel.
	<ol>
		<li>Average together duplicate wells for each standard, each sample and the Assay Blank for all 5 scans. Raw fluorescent values should increase across scans for the samples but remain stable (or decrease slightly) for standards and Assay Blanks.</li>
		<li>Take the highest AMC standard well average and divide by the known concentration (20 &#181;M). Divide this value by the sample concentration used in the assay to get standardized AMC value. For each sample and Assay Blank average, divide by the standardized AMC value.</li>
		<li>Take this final value and divide it by the sample concentration used in the assay to get the normalized value for each sample and the Assay Blank. Do this for all 5 scans.</li>
		<li>Subtract the normalized Assay Blank value from each normalized sample value for all 5 scans.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20S Proteasome Activity Kit</td>
			<td>Millipore Sigma</td>
			<td>APT280</td>
			<td>Other vendors carry different versions</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Fisher</td>
			<td>FERR1441</td>
			<td>Various other vendors</td>
		</tr>
		<tr>
			<td>BioTek Synergy H1 plate reader</td>
			<td>BioTek</td>
			<td>VATECHH1MT3</td>
			<td>Other vendors carry different versions</td>
		</tr>
		<tr>
			<td>Protease Inhibitor</td>
			<td>Millipore Sigma</td>
			<td>P8340</td>
			<td>Various other vendors</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>DMSO</td>
			<td>D8418</td>
			<td>Various other vendors</td>
		</tr>
	</tbody>
</table>

measuring proteasome activity in different subcellular compartments of the rat brain

Source: McFadden, T., et al. <a href="https://app.jove.com/v/59695">Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain.</a> J. Vis. Exp. (2019).
This video demonstrates how to measure proteasome activity in different subcellular compartments of the lateral amygdala from fear-conditioned and control rat brains using a fluorogenic peptide assay. The proteasome cleaves the fluorogenic peptide substrate, releasing fluorescent molecules, which are quantified using a plate reader to assess proteasome activity in the nuclear and synaptic protein fractions. Increased nuclear activity suggests changes in gene expression, while reduced synaptic activity indicates protein stabilization necessary for long-term memory maintenance.

Measuring Proteasome Activity in Different Subcellular Compartments of the Rat Brain

1. Proteasome Activity Assay
NOTE: Proteasome activity can be measured in homogenized brain tissue using a slightly modified version of the 20S Proteasome ...

Begin with subcellular nuclear and synaptic fractions collected from the lateral amygdala of the brains of both control and fear-conditioned rats.
Each subcellular fraction contains varying concentrations of 20S proteasomes, the catalytic core of the proteasome complex responsible for degrading unwanted cellular proteins.
Add an assay buffer containing ATP and a fluorogenic peptide substrate to the samples and incubate.
In the presence of ATP, the proteasome cleaves the fluorogenic peptide, releasing free fluorogenic molecules.
Using a plate reader, measure the fluorescence intensity at regular intervals.
Quantify the fluorescence by comparing it to known standard concentrations of the fluorogenic molecule.
The fluorescence intensity is proportional to the proteasome activity in the sample.
Increased proteasome activity in the nuclear fraction of fear-conditioned rats suggests changes in memory-related gene expression, while decreased activity in the synaptic fraction indicates changes in synaptic protein expression necessary for long-term memory maintenance.

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Research

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Neuroscience

Neuropathology

Neurodegenerative Diseases

41 Views. Source: McFadden, T., et al. Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain. J. Vis. Exp. (2019). This video demonstrates how to measure proteasome activity in different subcellular compartments of the lateral amygdala from fear-conditioned and control rat brains using a fluorogenic peptide assay. The proteasome cleaves the fluorogenic peptide substrate, releasing fluorescent molecules, which are quantified using a plate reader to assess proteasome activity in the nuc...

Video: Measuring Proteasome Activity in Different Subcellular Compartments of the Rat Brain - Concept

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric&#160;&#946; subunit rings sandwiched between two heptameric&#160;&#945; subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome &#945; and &#946; subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete &#945;-ring and one complete &#946;-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome assembly.

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

JoVE Journal

Biochemistry

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).

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

Biology

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.

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

Measuring Proteasome Activity in Different Subcellular Compartments of the Rat Brain

Transcript

Measuring Proteasome Activity in Different Subcellular Compartments of the Rat Brain

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Assays for the Degradation of Misfolded Proteins in Cells