Name: use of two dimensional semi-denaturing detergent agarose gel electrophoresis to confirm size heterogeneity of amyloid or amyloid-like fibers
Uploaded: 2018-04-26T14:30:00.000+00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers at JoVE.com

This work is supported by a fellowship for S.H.-A. from NIH/NCATS (TL1TR001104), a Welch Foundation grant (I-1827), and a R01 grant from NIGMS (RGM120502A) to Z.W. Z.W. is the Virginia Murchison Linthicum Scholar in Medical Research and Cancer Prevention and Research Institute of Texas Scholar (R1222).

1. Prepare Samples
<ol>
	<li>Culture 2 x&#160;106 amyloid producing HT-29 colon cancer cells in a 10-cm tissue culture dish in 10 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and penicillin-streptomycin. Culture cells overnight in a 37 &#176;C incubator with 5% CO2.
	<ol>
		<li>After the cells grow to 80% confluency, wash the cells with 10 mL of phosphate-buffered saline (PBS). Add 3 mL of Trypsin solution and incubate at 37 &#176;C for 3 min.</li>
		<li>After the cells are totally dissociated from the dish, add 10 mL of culture medium and transfer the cells with a 10-mL pipette to a 15-mL conical tube. Centrifuge the cells at 1,000 x g for 3 min at room temperature. Aspirate the medium, resuspend the cells in 5 mL of culture medium, and count the cells using a cell counter. Plate 2 x&#160;106 cells in each of two 10-cm dishes.</li>
		<li>Allow the cells to adhere and recover overnight in a 37 &#176;C incubator with 5% CO2. Apply treatment to one dish to induce the formation of amyloids with 20 ng/mL Tumor Necrosis Factor-Alpha (TNF-&#945;), 100 nM Smac-mimetic, and 20 &#181;M pan-caspase inhibitor Z-VAD-FMK11. The combination is abbreviated as TSZ. Treat the other dish with vehicle as a control.</li>
	</ol>
	</li>
	<li>After the appropriate length of time, usually 6 h, harvest the cell lysate.
	<ol>
		<li>Scrape the cells off the plate with a plastic scraper and use a 10-mL pipette to transfer into a 15-mL conical tube. Centrifuge the cells at 1,000 x g for 3 min at 4 &#176;C.</li>
		<li>Wash the cells 2 times by resuspending in 10 mL of ice cold PBS and centrifuging at 1,000 x g for 3 min at 4 &#176;C. Aspirate the PBS solution. 
		Note: The process can be paused here by freezing the cell pellet in liquid nitrogen and storing at &#727;80 &#176;C for up to 1 month.</li>
		<li>Transfer the cell pellet to a 1.5-mL microcentrifuge tube and incubate in 0.3 mL of lysis buffer for 30 min on ice. Centrifuge at 20,000 x g for 15 min at 4 &#176;C. The supernatant is the whole cell lysate. 
		Note: The process can be paused here by storing the sample at -20 &#176;C for up to several months.</li>
		<li>Measure the protein concentration by a Bradford assay according to manufacturer&#39;s protocol. Add 4x&#160;SDD-AGE loading buffer to prepare 20 &#181;L of 3 &#181;g/&#181;L sample and incubate at room temperature for 10 min.</li>
	</ol>
	</li>
</ol>
2. Prepare and Run Gels
<ol>
	<li>Add 2 g of agarose to 200 mL of 1x&#160;Tris-acetate buffer (TAE) in a glass beaker and heat in a microwave to melt the agarose. Add 1 mL of 20% SDS for a final concentration of 0.1% SDS. Carefully swirl to mix. Take care not to generate bubbles after the SDS addition.</li>
	<li>Pour the agarose solution into a 15 cm x&#160;14 cm gel slab. Use a 1-mL pipette to eliminate any bubbles. Place one 20-well comb at the top.</li>
	<li>First dimension: Pipette 60 &#181;g of whole cell lysate in the far-right lane. Run the gel at 60 V for about 4 h (until the dye front is about &#190; through the gel) using the TAE containing 0.1% SDS as the running buffer.</li>
	<li>Second dimension: Carefully rotate the gel 90&#176; counter-clockwise (Figure 2A). Run the gel at 60 V for about 4 h. 
	Note: The general running condition is 4 V/cm gel length. It is important that the running conditions are exactly the same for the first and second dimensions.</li>
</ol>
3. Transfer
<ol>
	<li>Use the capillary transfer method to transfer the proteins to a polyvinylidene difluoride (PVDF) membrane (Figure 2B).
	<ol>
		<li>Add 500 mL of transfer buffer to an approximately 20 cm x&#160;20 cm container. Make a 5-cm high stack of paper towels next to the container. The surface area of the top of the stack must be greater than the gel dimensions.</li>
		<li>Soak a 14 cm x&#160;15 cm filter paper in transfer buffer and place on top of the paper towel stack. Take care to place on the paper with no wrinkles or bubbles. Repeat with another piece of filter paper.</li>
		<li>Activate a 14 cm x&#160;15 cm PVDF membrane in methanol for 30 s then layer it on the filter paper taking care to use a roller to remove all bubbles. Rinse the gel off with transfer buffer and layer it on top of the membrane, again rolling out any bubbles.</li>
		<li>Cover the edge of the paper towels closest to the container of transfer buffer with plastic wrap. It is important that the paper bridge constructed in the next step does not come into direct contact with the paper towel stack.</li>
		<li>Soak a piece of 15 cm x&#160;35 cm filter paper in transfer buffer and place it so one end covers the top of the gel and the other end is in the container of transfer buffer. Repeat with another bridge.</li>
		<li>Cover the container with plastic wrap and leave overnight at room temperature.</li>
	</ol>
	</li>
</ol>
4. Western Blotting Detection
<ol>
	<li>Rinse the membrane with 50 mL of PBS containing 0.05% Tween-20 (PBST) in a 20 cm &#215; 20 cm container. Add 20 mL of 5% milk in PBST. Rock at room temperature for 30 min to block.</li>
	<li>Pipette 10 &#181;L of rabbit anti-MLKL antibody into 20 mL of 5% milk in PBST and add to the container. Incubate on rocker overnight at 4 &#176;C.</li>
	<li>Wash the membrane in 20 mL of PBST for 5 min. Repeat 5 times.</li>
	<li>Pipette 4 &#181;L of anti-rabbit-HRP to 20 mL of 5% milk in PBST and add to the container. Incubate on rocker at room temperature for 2 h.</li>
	<li>Wash the membrane in 20 mL of PBST for 5 min. Repeat 5 times.</li>
	<li>Add enhanced chemiluminescence substrate (ECL) and expose to X-ray films according to the manufacturer&#39;s protocol.</li>
</ol>

<ol>
	<li>Ross, C. A., Poirier, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+aggregation+and+neurodegenerative+disease.">Protein aggregation and neurodegenerative disease.</a> Nature medicine. 10, 10-17 (2004).</li><li>Hou, F., 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=MAVS+forms+functional+prion-like+aggregates+to+activate+and+propagate+antiviral+innate+immune+response.">MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.</a> Cell. 146, 448-461 (2011).</li><li>Li, J., 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+RIP1/RIP3+necrosome+forms+a+functional+amyloid+signaling+complex+required+for+programmed+necrosis.">The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.</a> Cell. 150, 339-350 (2012).</li><li>Liu, 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=MLKL+forms+disulfide+bond-dependent+amyloid-like+polymers+to+induce+necroptosis.">MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 114, 7450-7459 (2017).</li><li>Uptain, S. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+as+protein-based+genetic+elements.">Prions as protein-based genetic elements.</a> Annual review of microbiology. 56, 703-741 (2002).</li><li>Halfmann, R., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+amyloid+aggregation+by+Semi-Denaturing+Detergent-Agarose+Gel+Electrophoresis.">Screening for amyloid aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis.</a> J Vis Exp. , (2008).</li><li>Sipe, J. D., 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=Nomenclature+2014:+Amyloid+fibril+proteins+and+clinical+classification+of+the+amyloidosis.">Nomenclature 2014: Amyloid fibril proteins and clinical classification of the amyloidosis.</a> Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis. 21, 221-224 (2014).</li><li>Eisenberg, D. S., Sawaya, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Studies+of+Amyloid+Proteins+at+the+Molecular+Level.">Structural Studies of Amyloid Proteins at the Molecular Level.</a> Annual review of biochemistry. 86, 69-95 (2017).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Sun, L., 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=Mixed+lineage+kinase+domain-like+protein+mediates+necrosis+signaling+downstream+of+RIP3+kinase.">Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.</a> Cell. 148, 213-227 (2012).</li><li>He, 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=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137, 1100-1111 (2009).</li></ol>

The authors declare no conflict of interest.

The most critical aspect of the 2D SDD-AGE is that the electrophoresis conditions are the same for the first and second dimension. The ability to detect a sharp diagonal line at 45&#176; (indicating that no dissociation or degradation has occurred) depends on the fibers migrating in an identical manner during both electrophoreses. Using different conditions, for example changing the voltage or the length of run time, will obscure these results. Also, as is the case for traditional 1D SDD-AGE, boiling the sample should be...

The formation of amyloid fibers due to protein misfolding has long been known to play a role in pathologic conditions such as Alzheimer&#39;s disease, Parkinson&#39;s disease, and Huntington&#39;s disease1. More recently, the formation of amyloid or amyloid-like fibers has been shown to be a part of signaling pathways in humans, including during anti-viral innate immune response2 and necroptosis3,4, and in lower organisms such as yeast5,6. Therefore, the ability to detect these fibers in the lab is important. Currently, there are three main ways to detect amyloid and amyloid-like fibers: the use of dyes, EM, and SDD-AGE.
The use of dyes, such as Congo Red or Thioflavin T, offers the advantage of being rapid and easily detectable using either microscopy or spectroscopy7. However, detection by microscopy, in the case of Congo Red, provides no specificity about which proteins comprise the fibers, or the size of the fibers. Similarly, the use of spectroscopy to detect Congo Red or Thioflavin T binding to protein complexes provides only a positive or negative result.
EM provides conclusive evidence of the presence of fibers and also quantitative information about the fiber length and diameter8. However, this method requires very stringent purification. Additionally, EM is a specialized technique which uses expensive equipment.
SDD-AGE has been used to detect SDS-resistant mega Dalton protein complexes including amyloid or amyloid-like fibers. It offers many advantages. First, it does not require the purification of the fibers and is easy to peform9. Second, it provides qualitative information about the size of the fibers, including relative size and amount of fiber heterogenicity. Lastly, because Western blotting can be performed after electrophoresis, it is easy to detect the presence of any protein for which there is an antibody, although it should be noted that because SDD-AGE is semi-denaturing, some epitopes may remain concealed which complicates detection by antibody.
Recently, receptor interacting protein kinase 1 (RIPK1) and 3 (RIPK3) have been reported to form amyloid fibers to serve as signaling platforms during necroptosis, a programmed form of necrosis3. While studying these fibers, our lab showed that another necroptosis-associated protein, mixed lineage kinase domain-like (MLKL), also formed amyloid-like fibers4. However, upon examination with SDD-AGE, the size of the MLKL fibers appeared distinct from the RIPK1 and RIPK3 fibers, which appeared identical to each other (Figure 1). This was unexpected because it is well-known that MLKL binds to RIPK1/RIPK3 to form the necroptosis signaling complex called the necrosome10.
There are at least two explanations. First, two totally distinct amyloid-like fibers may form during necroptosis, one containing RIPK1/RIPK3 and the other containing MLKL. Second, only one type of amyloid-like fibers containing RIPK1/RIPK3/MLKL may be formed during necroptosis, but the association of MLKL with the other proteins is weak enough that it dissociates during SDD-AGE.
To address this, we propose performing a two-dimensional (2D) SDD-AGE. SDD-AGE-stable amyloid or amyloid-like fibers will have the same migration pattern during the first and second dimension electrophoresis. This will be detectable after transferring the proteins to a membrane and carrying out a Western blot. Stable fibers will exhibit a sharp diagonal pattern. Any deviation from this would suggest that the fibers undergo changes due to the SDS-electrophoresis.

<table><tbody><tr><td>gel electrophoresis unit</td><td>Fisher</td><td>HE99XPRO</td><td>appratus for gel running.</td></tr><tr><td>agrose</td><td>VWR</td><td>97062-250</td><td>For agarose gel.</td></tr><tr><td>paper tower</td><td>Fisher</td><td>19-120-2484</td><td>for transfering</td></tr><tr><td>filter paper</td><td>VWR</td><td>21427-386</td><td>for transfering</td></tr><tr><td>PVDF membrane</td><td>Bio-Rad</td><td>162-0177</td><td>for transfering</td></tr><tr><td>blocking milk powder</td><td>Bio-Rad</td><td>1706404XTU</td><td>for blocking in Western blot</td></tr><tr><td>MLKL antibody</td><td>Genetex</td><td>GTX107538</td><td>rabbit anti-MLKL antibody</td></tr><tr><td>RIPK1 antibody</td><td>BD Biosciences</td><td>610459</td><td>mouse anti-RIPK1 antibody</td></tr><tr><td>RIPK3 antibody</td><td>gift from Dr. Xiaodong Wang</td><td></td><td>rabbit anti-RIPK3. See refenence 11.</td></tr><tr><td>anti-Rabbit-HRP</td><td>Sigma</td><td>A6154</td><td>secondary antibody</td></tr><tr><td>ECL</td><td>Fisher</td><td>WBKLS0500</td><td>Western chemiluminescent HRP substrate</td></tr><tr><td>X-ray film</td><td>Fisher</td><td>F-BX810</td><td>for Western blot result</td></tr><tr><td>DMEM</td><td>Sigma</td><td>D6429</td><td>for cell culture</td></tr><tr><td>fetal bovine serum</td><td>Sigma</td><td>F4135</td><td>for cell culture</td></tr><tr><td>penicilin-streptomycin</td><td>Sigma</td><td>P4333</td><td>for cell culture</td></tr><tr><td>Trypsin solution</td><td>Sigma</td><td>T4049</td><td>for cell culture</td></tr><tr><td>PBS for tissue culture</td><td>Sigma</td><td>D8662</td><td>for cell culture</td></tr><tr><td>recombinant TNF</td><td>made in our lab</td><td></td><td>for inducing necroptosis. See reference 11.</td></tr><tr><td>smac-mimetic</td><td>gift from Dr. Xiaodong Wang</td><td></td><td>for inducing necroptosis. See reference 11.</td></tr><tr><td>ZVAD-FMK</td><td>ApexBio</td><td>A1902</td><td>for inducing necroptosis. See reference 11.</td></tr><tr><td>Cell Counter</td><td>Bio-Rad</td><td>1450102</td><td>Model TC20; for counting cells</td></tr><tr><td>Pierce&amp;trade; BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td>for measuring protein concentration in cell lysates</td></tr><tr><td>Cell lifter</td><td>Fisher</td><td>07-200-364</td><td>to remove cells from dish</td></tr><tr><td>Lysis Buffer (1 L)</td><td></td><td></td><td>20 mL 1 M Tris pH 7.4&lt;br/&gt; 10 mL glycerol&lt;br/&gt; 30 mL 5 M NaCl&lt;br/&gt; 840 mL ddH&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 10 mL Triton-X100&lt;br/&gt; (protease and phosphates inhibitors as desired)</td></tr><tr><td>10X TAE (1 L)</td><td></td><td></td><td>48.4 g Tris base&lt;br/&gt; 11.42 mL glacial acetic acid&lt;br/&gt; 20 mL 0.5M EDTA pH 8&lt;br/&gt; ddH&lt;sub&gt;2&lt;/sub&gt;0 to 1 L</td></tr><tr><td>4X SDD-AGE loading buffer (50 mL)</td><td></td><td></td><td>5 mL 10X TAE&lt;br/&gt; 10 mL glycerol&lt;br/&gt; 4 mL 20% SDS&lt;br/&gt; 0.5 mL 10% bromophenol blue&lt;br/&gt; 31 mL ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>TBS Transfer Buffer (1 L)</td><td></td><td></td><td>20 mL 1 M Tris pH 7.4&lt;br/&gt; 30 mL 5 M NaCl&lt;br/&gt; 950 mL ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>PBST Wash Buffer (1 L)</td><td></td><td></td><td>100 mL 10xPBS&lt;br/&gt; 800 mL ddH&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 1 mL Tween20</td></tr><tr><td>10X PBS (10 L)</td><td></td><td></td><td>800 g NaCl&lt;br/&gt; 20 g KCl&lt;br/&gt; 144 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 24 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; add ddH&lt;sub&gt;2&lt;/sub&gt;O to 10 L</td></tr></tbody></table>

use of two dimensional semi-denaturing detergent agarose gel electrophoresis to confirm size heterogeneity of amyloid or amyloid-like fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

After necroptosis induction, RIPK1 and RIPK3 showed almost identical amyloid-like patterns (lanes 2 and 4, Figure 1). However, MLKL fibers were more heterogeneous and seemed to be smaller than RIPK1/RIPK3 fibers (lane 6, Figure 1). That prompted us to develop the 2D SDD-AGE method to address the possibility whether MLKL forms RIPK1/RIPK3-independent fibers or MLKL simply dissociates from the large RIPK1/RIPK3/MLKL fibers during S...

Watch this Scientific Journal Video about Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers at JoVE.com

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

use-two-dimensional-semi-denaturing-detergent-agarose-gel

Research

JoVE Journal

Biochemistry

9.7K Views.  UT Southwestern Medical Center. Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Video: Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

Neuroscience

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Biology

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Characterizing Spherulites with Polarization-Sensitive Two-Photon Microscopy

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

Author Spotlight: Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy 

This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid fibrils.

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue ...

Chemistry

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 ...

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight

Source: Sultana, S. et al. <a href="https://www.jove.com/v/62628">Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor</a>. J. Vis. Exp. (2021).
This video demonstrates a denaturing polyacrylamide-based separation technique for proteins. This technique helps in the preliminary identification of proteins based on their molecular weights.

Source: Sultana, S. et al. Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor. J. Vis. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Isolation of Amyloid Fibrils from Brain Tissue Extract

Source: Upadhyay, A. et al., <a href="https://app.jove.com/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

Neuropathology

Neurodegenerative Diseases

Two-Dimensional Semi-Denaturing Agarose Gel Electrophoresis: A Technique to Separate Polymorphic Amyloids Fibers Based on Size Heterogeneity

Source: Hanna-Addams, S., et al. <a href="https://www.jove.com/v/57498">Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers</a>. J. Vis. Exp. (2018).
This video demonstrates the two-dimensional semi-denaturing detergent agarose gel electrophoresis-based separation of amyloid fibers. The technique separates the polymorphic amyloid aggregates based on their heterogeneous size.

Source: Hanna-Addams, S., et al. Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or ...

Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

Source: Zhan, X., et al. <a href="https://www.jove.com/v/56739">Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome</a> J. Vis. Exp. (2018)
In this video, we demonstrate a two-dimensional gel electrophoresis method that separates individual proteins from a complex protein mixture obtained from tumor tissues of human nonfunctional pituitary adenoma (NFPA). The proteins are separated based on their electric charge in the first dimension and further separated based on molecular weights in the second dimension.

Source: Zhan, X., et al. Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Summary

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SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

A11-positive β-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis