The authors thank Drs. Jim Lah and Allan Levey, Emory Department of Neurology, for helpful comments and suggestions. This work was partly funded by the Accelerating Medicine Partnership grant (U01AG046161-02), the Emory Alzheimer&#39;s Disease Research Center (P50AG025688) and a National Institute on Aging grant (R01AG053960-01) to N.T.S. This research was also supported in part by the Neuropathology Core of the Emory Neuroscience NINDS Core Facilities grant, P30NS055077.

Ethics Statement: All brain tissues were obtained from the Emory Alzheimer&#39;s Disease Research Center (ADRC) Brain Bank. Human postmortem tissues were acquired under proper Institutional Review Board (IRB) protocols.
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 from the Emory ADRC brain bank (n=2). Post-mortem neuro...

<ol>
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Biol. Chem. 284 (47), 32895-32905 (2009).</li><li>Bai, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+small+nuclear+ribonucleoprotein+complex+and+RNA+splicing+alterations+in+Alzheimer's+disease.">U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.</a> Proc. Natl. Acad. Sci. 110 (41), 16562-16567 (2013).</li><li>Klunk, W. E., Pettegrew, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+&#946;-Amyloid+Protein+Is+Covalently+Modified+when+Dissolved+in+Formic+Acid.">Alzheimer's &#946;-Amyloid Protein Is Covalently Modified when Dissolved in Formic Acid.</a> J Neurochem. 54 (6), 2050-2056 (1990).</li><li>Yang, L. -. S., Gordon-Krajcer, W., Ksiezak-Reding, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+Released+from+Paired+Helical+Filaments+with+Formic+Acid+or+Guanidine+Is+Susceptible+to+Calpain-Mediated+Proteolysis.">Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis.</a> J Neurochem. 69 (4), 1548-1558 (1997).</li><li>Bai, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+small+nuclear+ribonucleoprotein+complex+and+RNA+splicing+alterations+in+Alzheimer's+disease.">U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 110 (41), 16562-16567 (2013).</li><li>Hales, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+small+nuclear+ribonucleoproteins+(snRNPs)+aggregate+in+Alzheimer's+disease+due+to+autosomal+dominant+genetic+mutations+and+trisomy+21.">U1 small nuclear ribonucleoproteins (snRNPs) aggregate in Alzheimer's disease due to autosomal dominant genetic mutations and trisomy 21.</a> Mol Neurodegener. 9 (1), 15 (2014).</li><li>Xiong, L. -. W., Raymond, L. D., Hayes, S. F., Raymond, G. J., Caughey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+change,+aggregation+and+fibril+formation+induced+by+detergent+treatments+of+cellular+prion+protein.">Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein.</a> J Neurochem. 79 (3), 669-678 (2001).</li><li>Mirra, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Consortium+to+Establish+a+Registry+for+Alzheimer's+Disease+(CERAD).+Part+II.+Standardization+of+the+neuropathologic+assessment+of+Alzheimer's+disease.">The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease.</a> Neurology. 41 (4), 479-486 (1991).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150 (1), 76-85 (1985).</li><li>Guo, J. 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The authors have nothing to disclose.

Herein we introduce and discuss an abbreviated single-step detergent-fractionation protocol that is applicable to a wide variety of experimental applications ranging from mass spectrometry-based proteomics analysis to functional protein misfolding assays and western blotting.5,6,7,10 This methodology is perhaps most effective when used to study neurodegenerative proteinopathies such as Alzheime...

Neurodegenerative diseases such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), amyotrophic lateral sclerosis (ALS), and the closely related prion diseases are proteinopathies characterized by the gradual accumulation of detergent-insoluble protein aggregates in the brain with accompanying cognitive decline.1,2 This shared pathological feature is thought to play a central role in the etiology and pathophysiology of these neurodegenerative diseases.2 These aggregates typically consist of polymeric amyloid fibers, which are composed of repeating units of misfolded protein exhibiting cross &#946;-structure.1,2,3,4 Biochemically, amyloid aggregates are highly resistant to chemical or thermal denaturation and solubilization,3 which presents unique challenges to their purification, analysis and study via traditional biochemical techniques.2,5,6,7,8,9,10,11 Unsurprisingly, the detergent-insoluble protein fraction has been the focus of much research into the pathophysiology of neurodegenerative diseases involving the accumulation of misfolded proteins.6,12,13,14
Biochemical fractionation techniques have often been utilized to enrich the detergent-insoluble fraction from postmortem brain homogenates.6,12,13,14 One of the most common methods involves the sequential extraction of tissue homogenates with buffers and detergents of increasing stringency, followed by ultracentrifugation to partition the soluble and insoluble fractions. A commonly used sequential fractionation protocol6,14 involves the homogenization of frozen tissue samples in a detergent-free low salt (LS) buffer and the resultant insoluble pellets are then sequentially extracted with buffers containing high salt, non-ionic detergents, high sucrose, ionic detergents and finally chaotropes like urea.6,14 An obvious drawback of such a sequential fractionation protocols is the substantial time and labor commitment required to complete them. Including homogenization and ultracentrifugation, a typical five-step fractionation protocol can take several hours or even days to complete. 4,6,7,10,15,16,17,18 Additionally, as many pathologic protein aggregates remain insoluble in all but the harshest conditions19,20 most of the generated fractions are of limited value. Thus, the less-stringent fractionation steps utilizing high salt concentrations and non-ionic detergents are largely redundant.
Previous studies have shown that the ionic detergent N-lauryl-sarcosine (sarkosyl) is an excellent candidate for a simplified single-step detergent fractionation protocol.5,6,12,13,14,21,22,23 As a denaturing detergent, sarkosyl is stringent enough to solubilize the vast majority of natively folded proteins in brain without solubilizing misfolded protein aggregates composed of beta-amyloid (A&#946;),6,11 phosphorylated tau (pTau),6 TAR DNA-binding protein 43 (TDP-43),14 alpha-synuclein,12,13 scrapie,23 or U1 small nuclear ribonucleoproteins (U1 snRNPs) such as U1-70K.5,21,22As sarkosyl is less stringent than the ubiquitous anionic detergent sodium dodecyl sulfate (SDS), it preserves less robust oligomeric forms of misfolded protein aggregates that cannot withstand SDS treatment.9
Previously, we described an abbreviated detergent-fractionation protocol that achieved results comparable to the more laborious sequential fractionation methodologies.5 By omitting the less stringent fractionation steps, we were able to develop a facile single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from postmortem human brain.5 This detailed protocol described herein is well suited for a wide range of experimental applications ranging from Western blotting and mass spectrometry-based proteomics to functional protein misfolding and aggregation seeding assays.5,6,21

<table>
	<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>4X SDS sample buffer</td>
			<td>Home-made</td>
			<td>N/A</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>TCEP solution, neutral pH</td>
			<td>Thermo Fisher</td>
			<td>77720</td>
			<td>reducing agent</td>
		</tr>
		<tr>
			<td>(TBS) blocking buffer</td>
			<td>Thermo Fisher</td>
			<td>37542</td>
			<td>blocking buffer</td>
		</tr>
		<tr>
			<td>(TBS) blocking buffer + 0.05% Tween 20</td>
			<td>Thermo Fisher</td>
			<td>37543</td>
			<td>blocking buffer and antibody diluent</td>
		</tr>
		<tr>
			<td>4-12% Bolt Bis-Tris Plus gels, 10-well</td>
			<td>Thermo Fisher</td>
			<td>NW04120BOX</td>
			<td>precast SDS-PAGE gels</td>
		</tr>
		<tr>
			<td>MES SDS Running Buffer (20X)</td>
			<td>Thermo Fisher</td>
			<td>B0002</td>
			<td>SDS-PAGE running buffer</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>
		<tr>
			<td>Anti-Tau-2 (pan tau) antibody</td>
			<td>Chemicon</td>
			<td>MAB375</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-phospho-threonine 231 Tau antibody</td>
			<td>Millipore</td>
			<td>MAB5450</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8)</td>
			<td>Thermo Fisher</td>
			<td>MN1020</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-early endosome antigen 1 (EEA1) antibody</td>
			<td>abcam</td>
			<td>ab2900</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher</td>
			<td>A21058</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher</td>
			<td>A11374</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>iBlot2 Dry Blotting System</td>
			<td>Thermo Fisher</td>
			<td>IB21001</td>
			<td>Gel transfer</td>
		</tr>
		<tr>
			<td>iBlot2 Transfer Stacks, Nitrocellulose, mini</td>
			<td>Thermo Fisher</td>
			<td>IB23002</td>
			<td>Gel transfer</td>
		</tr>
	</tbody>
</table>

enrichment of detergent-insoluble protein aggregates from human postmortem brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

The abbreviated single-step sarkosyl-fractionation protocol was used to enrich detergent-insoluble protein aggregates from control and AD postmortem brain (Figure 1). Proteins from TH-S, S1, S2 and P2 fractions were resolved by SDS-PAGE, fixed for 15 min in Coomassie blue fixative buffer followed by gentle staining with Coomassie Brilliant Blue G-250 staining buffer. The resuspension step is optional since there were undetectable levels of protein in the S2 f...

Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

The overall goal of this fractionation protocol is to enrich detergent-insoluble protein aggregates from human postmortem brain tissue. So this method can help answer key questions in the field of neurodegeneration, such as the identification and characterization of misfolded protein aggregates in disease. The main advantage of this technique is the fascial enrichment and isolation of detergent-insoluble protein aggregates from brain tissue within a short time frame.Demonstrating this procedure will be Tram Nguyen, a technician from my laboratory. 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 prechilled Dounce homogenizer tube on ice. Remember to avoid white matter and any large blood vessels.Work quickly and place the tissue on TARE 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 minus 80 degrees Celsius. To proceed with fractionation, add 100 microliters of five molar sodium chloride and 100 microliters of 10 percent sarkosyl to each 0.8-milliliter aliquot.Mix well by inversion. And then incubate on ice for 15 minutes. Following the incubation, use a sonicator equipped with a microtip probe to deliver three five-second pulses at 30 percent 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 milliliter. Then transfer 500 microliters of each sample into 500-microliter polycarbonate ultracentrifuge tubes. Load tubes into a prechilled rotor and ultracentrifuge at 180, 000 times g for 30 minutes at four degrees Celsius.Following the centrifugation, transfer the S1 sarkosyl-soluble supernatants to 1.5-milliliter tubes and store at minus 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 re-suspended P1 pellets to new 500-microliter ultracentrifuge tubes, pair-balance, and centrifuge at 180, 000 times g for an additional 30 minutes at four 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 re-suspended P2 pellets to labeled 0.5-milliliter tubes and fully solubilize the pellets, using a brief one-second microtip sonication at 20 percent amplitude.Determine the protein concentrations of the sarkosyl-soluble and sarkosyl-insoluble fractions using the BCA assay method. Prepare 40-microgram samples of total homogenate sark-soluble S1 and sark-insoluble P2 fractions in one-x Laemmli SDS-PAGE sample buffer with five millimolar TCEP at pH seven. Incubate samples at 95 degrees Celsius for five minutes.Load the samples onto precast 10-well four to 12 percent Bis-Tris SDS-PAGE gels. Electrophorese at 80 volts for the first centimeter and 120 volts for about an hour or until the tracking dye reaches the bottom of the gel. Submerge the precast gel cassettes into distilled water and then use a fresh razor blade and gel knife to split open.Gently cut away the combs and very bottom of each gel with a fresh razor blade. Transfer to 0.2 micron nitrocellulose membranes using a dry transfer method on a blotting machine. After transfer, block the membranes in blocking buffer without Tween-20 for 30 minutes, followed with blocking buffer with 0.5 percent Tween-20 for 30 minutes.After blocking, rinse the membranes in TBS with 0.1 percent Tween-20 for five minutes to remove excess blocking buffer. Prepare 1:1, 000 dilutions of the primary antibodies in blocking buffer with 0.5 percent Tween-20. Phospho-Tau pT231 and EEA1 are used here.Incubate the membranes in primary antibody solution overnight at four degrees Celsius with circular agitation. The next day, rinse the membranes in TBS with 0.1 percent Tween-20 three times for 15 minutes each. Probe the membranes with a 1:20, 000 dilution of the fluorescently-labeled near-infrared secondary antibody and blocking buffer with 0.5 percent Tween-20 for 60 minutes at room temperature in darkness on a shaker.Following the incubation, rinse the membrane three times for 10 minutes in TBS with 0.1 percent Tween-20 and twice in TBS for five minutes each time. Scan the membrane using an infrared imaging system at the appropriate excitation wavelength for the secondary antibody used. This Coomassie-stained gel shows 20 mcg of protein from TH-S, S1, S2, and P2 fractions.Western blot Analysis clearly demonstrates that in AD brain, the pathologic high-molecular weight phosphorylated Tau aggregates with sarkosyl-insoluble and very little phospho-Tau remained in the sarkosyl fractions. EEA1 was used as a general marker for most native nonpathologic proteins that are inherently sarkosyl-soluble and partitioned into the S1 fraction in both control and AD brain. Notably, there was slightly less signal for EEA1 in the soluble fraction compared to the TH-S fraction, indicating that a minor pool of EEA1 is likely sarkosyl-insoluble, yet below the limit of detection by western blot with this particular antibody.This figure shows the proteomic analysis of pathologic AD proteins using a single-step or multi-step fractionation protocol. The single-step fractionation technique may be advantageous for low-abundance proteins, as the overall sample losses are significantly reduced. While attempting this procedure, it's important to remember to excise as much white matter from the brain tissue as possible.Following this procedure, other methods like mass-spectrometry-based proteomics can be performed in order to identify and characterize core protein components of pathological aggregates in their degenerative diseases.

enrichment-detergent-insoluble-protein-aggregates-from-human

Research

JoVE Journal

Biochemistry

11.8K Views.  Emory School of Medicine. The overall goal of this fractionation protocol is to enrich detergent-insoluble protein aggregates from human postmortem brain tissue. So this method can help answer key questions in the field of neurodegeneration, such as the identification and characterization of misfolded protein aggregates in disease. The main advantage of this technique is the fascial enrichment and isolation of detergent-insoluble protein aggregates from brain tissue within a short time frame.Demonstrating this ...