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

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

Obtain frozen postmortem brain tissue from healthy control and pathologically confirmed AD. Use forceps and a razor blade to excise approximately 250 milligram portions of gray matter from each tissue sample. Allow the brain segment to thaw for one minute. Now, dice a piece of tissue into roughly two-millimeter cubed pieces as it thaws. Transfer to a two-milliliter pre-chilled dounce homogenizer tube on ice.
Remember to avoid white matter and any large blood vessels.
Work quickly and place the tissue on tear disposable weigh boats. Take care to prevent the tissue from thawing by frequently placing the weighing boat with brain tissue into polystyrene containers with dry ice. After dissecting and weighing all required pieces of tissue, remove a weigh boat containing a piece of tissue from dry ice.
Next, add five milliliters of ice cold low-salt buffer with protease and phosphatase inhibitor cocktail per gram of tissue, and homogenize on ice using approximately 10 strokes of high-clearance pestle A and 15 strokes of low-clearance pestle B. Then, use a nine-inch glass Pasteur pipette to transfer each homogenate to a labeled two-milliliter polypropylene tube. Aliquot the remaining homogenate into additional labeled 1.5-milliliter tubes in a similar fashion for storage at -80 degrees Celsius.
To proceed with fractionation, add 100 microliters of five molar sodium chloride and 100 microliters of 10% Sarkosyl to each 0.8-millimeter aliquot. Mix well by inversion, and then, incubate on ice for 15 minutes. Following the incubation, use a sonicator equipped with a micro-tip probe to deliver three 5-second pulses at 30% amplitude.
After using the bicinchoninic acid method to determine the protein concentrations of the homogenates, add ice-cold sark buffer with protease and phosphatase inhibitors to each sample to a final concentration of 10 milligrams per millimeter. Then, transfer 500 microliters of each sample into 500 microliter polycarbonate ultracentrifuge tubes.
Load tubes into a pre-chilled rotor, and ultracentrifuge at 180,000 times g for 30 minutes at 4 degrees Celsius. Following the centrifugation, transfer the S1 sarkosyl soluble supernatants to 1.5-milliliter tubes and store at -80 degrees Celsius. Add 200 microliters of sark buffer to the ultracentrifuge tubes containing the detergent insoluble P1 fractions and use the pipette tip to dislodge the pellets from the bottom of the ultracentrifuge tubes.
Next, transfer the resuspended P1 pellets to new 500-microliter ultracentrifuge tubes, pair balance, and centrifuge at 180,000 times g for an additional 30 minutes at 4 degrees Celsius. Discard the S2 supernatant, then add 50 to 75 microliters of urea buffer with protease and phosphatase inhibitor cocktail to the sarkosyl insoluble pellets and incubate for 30 minutes at room temperature to solubilize the P2 pellet.
After the incubation, transfer the resuspended P2 pellets to labeled 0.5-millimeter tubes and fully solubilize the pellets using a brief 1 second microtip sonication at 20% amplitude. Determine the protein concentrations of the sarkosyl soluble and sarkosyl insoluble fractions using the BCA assay method.

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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>

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.

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;

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

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