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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.&#160;&#160;
1. Homogenization and Fractionation
<ol>
	<li>Tissue selection 
	NOTE: Frozen postmortem frontal cortex tissue from healthy control (Ctl) and pathologically confirmed AD cases were selected (n=2). Post-mortem neuropathological evaluation of amyloid plaque distribution was performed according to the Consortium to Establish a Registry for Alzheimer&#39;s Disease semi-quantitative scoring criteria, although neurofibrillary tangle pathology was assessed in accordance with the Braak staging system.
	<ol>
		<li>Obtain frozen postmortem brain tissue from healthy control (Ctl) and pathologically confirmed AD cases. Utilizing containers of dry ice, forceps and a razor blade, excise ~ 250 mg portions of grey matter from each tissue sample on tared disposable weigh boats, taking care to prevent the tissue from thawing. Record the weight of each piece to be homogenized.</li>
		<li>Excise ~ 250 mg of grey matter using forceps and razor by visually inspecting to locate the interface between the outer grey matter and inner white matter. Avoid white matter and more importantly, any large blood vessels or bloody regions, meninges, or arachnoid mater. Keep the tissue frozen during the cutting process by working quickly and frequently placing weighing boat with brain tissue into polystyrene containers with dry ice.</li>
		<li>Record the exact weight of grey matter excised from each frozen piece using an analytical balance. Calculate the volume of low salt (LS) buffer (see Table 1) needed for dounce homogenization (5 mL/g or 20% w/v).</li>
		<li>Prepare 10 mL LS buffer with 1x protease and phosphatase inhibitor cocktail and chill on ice.</li>
		<li>Remove weighed tissue and weighing boat from dry ice and dice into roughly 2 x 2 mm pieces as it thaws. Transfer to a 2 mL pre-chilled dounce homogenizer tube on ice.</li>
		<li>Add 5 mL/g (20% w/v) of ice-cold low salt (LS) buffer with 1X protease and phosphatase inhibitor cocktail.</li>
		<li>Homogenize the brain tissue on ice using approximately 10 strokes of high clearance pestle A and 15 strokes of low clearance pestle B.</li>
		<li>Transfer all homogenate to a 2 mL polypropylene tube using a glass Pasteur pipette. Label the tube with &#34;TH&#34; (Total Homogenate), the tissue case number, and homogenate volume.&#160; Aliquot 0.8 mL into a labeled 1.5 mL tube. Any excess homogenate can be similarly aliquoted and stored at -80 &#176;C.</li>
		<li>To each 0.8 mL aliquot, add 100 &#181;L each of 5 M NaCl and 10% (w/v) sarkosyl to concentrations of 0.5 M and 1% w/v, respectively. Mix tubes well by inversion and incubate on ice for 15 min. Label tubes with &#34;TH-S&#34; (Total Homogenate-Sarkosyl) to indicate that the homogenates are in sarkosyl-buffer (Table 1).</li>
		<li>Sonicate each tube for three 5 s pulses at 30% amplitude on ice (maximum intensity = 40%) using a microtip probe.</li>
		<li>Determine the protein concentrations of the homogenates using the bicinchoninic acid (BCA) assay method. 
		NOTE: The average protein concentration of TH-S homogenates prepared at 5 mL LS per gram of tissue is 15-20 mg/mL. The homogenates can be fractionated immediately or stored at -80 &#176;C until use.</li>
		<li>Dilute TH-S homogenates to 10 mg/mL using ice-cold sark buffer (Table 1) with 1x protease and phosphatase inhibitors.</li>
		<li>Transfer 5 mg protein (0.5 mL) of each TH-S homogenate into 500 &#181;L polycarbonate ultracentrifuge tubes and pair-balance with sark-buffer. Load tubes into a pre-chilled rotor and ultracentrifuge at 180,000 x g for 30 min at 4 &#176;C. Transfer the sarkosyl-soluble supernatants (S1) to 1.5 mL tubes and store at -80 &#176;C. 
		NOTE: (Optional wash) Add 200 &#181;L of sark-buffer to the ultracentrifuge tubes containing the detergent-insoluble fractions (P1) and dislodge the pellets (P1) from the bottom of the ultracentrifuge tubes using a 200 &#181;L pipette tip.</li>
		<li>Briefly pulse-spin the inverted tubes for 2-3 s (&#8804;2,500 x g) on a microcentrifuge to transfer the pellets (P1) and buffer to the 1.5 mL tubes below. Resuspend the insoluble pellets (P1) in the sark-buffer by pipetting up and down to ensure the pellet is disrupted.</li>
		<li>Pair-balance, and centrifuge at 180,000 x g for an additional 30 min at 4 &#176;C.</li>
		<li>Discard the optional wash supernatant (S2) and incubate the sarkosyl-insoluble pellets (P2) in 50-75 &#181;L of urea buffer (Table 1) with 1x PIC (protease and phosphatase inhibitor cocktail) for 30 min at room temperature to solubilize the pellet. 
		NOTE: Warm urea buffer to room temperature before use to avoid SDS precipitation. For detergent-sensitive applications, omit SDS from urea buffer and consider washing the insoluble pellet (P2) in low salt buffer before resuspending in urea buffer.</li>
		<li>Transfer the resuspended pellets (P2) to 0.5 mL tubes and use brief (1 s) microtip sonication at 20% amplitude (maximum intensity = 40%) to fully solubilize the pellets.</li>
		<li>Determine the protein concentrations of the sarkosyl-soluble (S1) and -insoluble (P2) fractions using the BCA assay method. Use these fractions immediately or store at -80 &#176;C until use.</li>
	</ol>
	</li>
</ol>
Table 1: Buffers list
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% w/v sarkosyl solution: 10 g N-lauryl-sarcosine sodium salt per 100 ml of ddH2O (stir at RT overnight)</td>
		</tr>
		<tr>
			<td>Low Salt (LS) buffer: 50 mM HEPES pH 7.0, 250 mM sucrose, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>Sarkosyl (sark) buffer: LS buffer + 1% (w/v) sarkosyl + 0.5 M NaCl</td>
		</tr>
		<tr>
			<td>Urea buffer (store at -20 &#176;C): 50 mM Tris-HCl pH 8.5, 8M urea, 2% SDS</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protease and phosphatase inhibitor cocktail, EDTA-free (100X)</td>
			<td>Thermo Fisher</td>
			<td>78441</td>
			<td>protease &#38; phosphatase inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator System (ultrasonicator)</td>
			<td>Fisher Scientific</td>
			<td>FB505110</td>
			<td>microtip ultrasonicator</td>
		</tr>
		<tr>
			<td>Optimax TLX Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>361545</td>
			<td>refrigerated ultracentrifuge</td>
		</tr>
		<tr>
			<td>TLA120.1 rotor</td>
			<td>Beckman Coulter</td>
			<td>362224</td>
			<td>ultracentrifuge rotor</td>
		</tr>
		<tr>
			<td>500 ul (8x34mm) polycarbonate tubes, thickwall</td>
			<td>Beckman Coulter</td>
			<td>343776</td>
			<td>ultracentrifuge tubes for TLA120.1 rotor</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt (sarkosyl)</td>
			<td>Sigma Aldrich</td>
			<td>L5777-50G</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>1.5 ml polypropylene Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749521-1500</td>
			<td>homogenization tool</td>
		</tr>
		<tr>
			<td>Cordless motor for Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749540-0000</td>
			<td>homogenization tool</td>
		</tr>
	</tbody>
</table>

isolation of detergent-insoluble protein aggregates from human postmortem brain tissue

Source: Diner, I., et al. <a href="https://app.jove.com/v/55835">Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain.</a> J. Vis. Exp. (2017).
This video explains the process of isolating and enriching detergent-insoluble protein aggregates from frozen postmortem human brain tissue. It demonstrates key steps, including homogenization, detergent treatment, sonication, and ultracentrifugation to separate and purify the insoluble aggregates. The final step involves solubilizing the aggregates in urea buffer for further analysis.

Isolation of Detergent-Insoluble Protein Aggregates From Human Postmortem Brain Tissue

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.&#160;&#160;
1. Homogenization and ...

Begin with a frozen postmortem human brain tissue sample from a confirmed neurodegenerative disease case.
Excise grey matter rich in insoluble protein aggregates, a hallmark of the disease.
Transfer the tissue to a homogenizer.
Add a buffer containing inhibitors and homogenize to release intracellular components.
The inhibitors block protease and phosphatase enzymes, preventing protein degradation.
Transfer the homogenate to a tube and add an ionic detergent buffer. Incubate.
The detergent solubilizes natively folded proteins, while misfolded protein aggregates remain intact.
Sonicate to complete cellular dissociation.
Transfer the homogenate to an ultracentrifuge tube and centrifuge at high speed.
The aggregates settle at the bottom.
Remove the supernatant containing detergent-soluble proteins.
Wash the aggregates with an ionic detergent buffer, centrifuge, and remove the supernatant.
Add urea buffer to denature the protein aggregates, making them soluble.
Transfer the solubilized protein aggregates to a tube and sonicate briefly to ensure complete dissolution.&#160;&#160;

isolation-detergent-insoluble-protein-aggregates-from-human

Research

JoVE Encyclopedia of Experiments

Neuroscience

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Neurodegenerative Diseases

29 Views. Źródło: Diner, I., et al. Wzbogacenie nierozpuszczalnych w detergentach agregatów białkowych z ludzkiego mózgu pośmiertnego. J. Vis. Exp. (2017). Ten film wyjaśnia proces izolowania i wzbogacania nierozpuszczalnych w detergentach agregatów białkowych z zamrożonej pośmiertnej ludzkiej tkanki mózgowej. Demonstruje kluczowe etapy, w tym homogenizację, obróbkę detergentami, sonikację i ultrawirowanie w celu oddzielenia i oczyszczenia nierozpuszczalnych agregatów. Ostatnim etapem j...

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The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
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For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

JoVE Journal

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Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

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Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

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Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Isolation of Detergent-Insoluble Protein Aggregates From Human Postmortem Brain Tissue

Transkrypcja

Isolation of Detergent-Insoluble Protein Aggregates From Human Postmortem Brain Tissue