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 avoided, as this will disrupt amyloid and amyloid-like fibers. As is the case for all transfers, care should be taken to avoid bubbles between the layers of filter paper, membrane, and gel. Lastly, during the transfer step, it is critical that the bridge does not droop and directly contact the paper towel stack. A reliable way to prevent this is to use a small piece of plastic wrap to cover the edge of the paper towels.
The running conditions provided in the protocol (60 V for 4 h) may be adjusted. For example, in this protocol, the agarose gel was 14 cm x&#160;15 cm. If a smaller gel is used, voltage (4 V/cm gel length) and run time should be adjusted proportionally. Two samples may be analyzed at once by using two combs when preparing the gel. The samples should be loaded in the right most well for each comb. In this case, the run time should again be shortened so that the top sample does not run into the bottom half of the gel. If the time is adjusted for the one dimension of the gel, it is critical that the other dimension is adjusted to match.
In contrast to SDS-PAGE, both 1D and 2D SDD-AGE are semi-denaturing. Because the fibers remain intact, some epitopes might remain inaccessible. This may limit detection by some antibodies. 2D SDD-AGE is limited in its ability to determine the exact size of amyloid or amyloid-like fibers. However, a relative size comparison may be made.
All protein complexes are subject to dissociation or degradation during SDD-AGE, and so the qualitative results may be difficult to interpret. The use of 2D SDD-AGE permits confirmation that size heterogeneity seen during SDD-AGE is not due to SDS-dependent dissociation during electrophoresis. 2D SDD-AGE is appropriate to use in any context where traditional 1D SDD-AGE is used. 2D SDD-AGE is especially useful when size heterogeneity is observed in 1D SDD-AGE.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8482; 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 
			10 mL glycerol 
			30 mL 5 M NaCl 
			840 mL ddH2O 
			10 mL Triton-X100 
			(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 
			11.42 mL glacial acetic acid 
			20 mL 0.5M EDTA pH 8 
			ddH20 to 1 L</td>
		</tr>
		<tr >
			<td >4X SDD-AGE loading buffer (50 mL)</td>
			<td></td>
			<td></td>
			<td >5 mL 10X TAE 
			10 mL glycerol 
			4 mL 20% SDS 
			0.5 mL 10% bromophenol blue 
			31 mL ddH2O</td>
		</tr>
		<tr >
			<td >TBS Transfer Buffer (1 L)</td>
			<td></td>
			<td></td>
			<td >20 mL 1 M Tris pH 7.4 
			30 mL 5 M NaCl 
			950 mL ddH2O</td>
		</tr>
		<tr >
			<td >PBST Wash Buffer (1 L)</td>
			<td></td>
			<td></td>
			<td >100 mL 10xPBS 
			800 mL ddH2O 
			1 mL Tween20</td>
		</tr>
		<tr >
			<td >10X PBS (10 L)</td>
			<td></td>
			<td></td>
			<td >800 g NaCl 
			20 g KCl 
			144 g Na2HPO4&#183;2H2O 
			24 g KH2PO4 
			add ddH2O 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 SDD-AGE.
A general schematic of the electrophoresis and transfer procedure is shown in Figure 2. During the first dimension SDD-AGE, amyloid or amyloid-like fibers will exhibit a characteristic smear, as seen in Figure 3A. Upon running the second dimension SDD-AGE, there are two possible outcomes. In the case of no degradation or dissociation during the gel running process, the fibers will migrate identically during the second run as they did in the first run. In this case, a Western blot will exhibit a diagonal pattern on the membrane at a 45&#176; angle as seen in Figure 3B. If the fibers dissociate during the SDD-AGE process, the smaller fibers will migrate faster during the second electrophoresis. In this case, a Western blot will exhibit vertical streaking below the diagonal line, as shown in Figure 3C.
In the case of the MLKL fibers seen during necroptosis, they show the characteristic smear during the first dimension SDD-AGE (Figure 4A). When those same fibers were subjected to 2D SDD-AGE, they exhibited a sharp diagonal line with no vertical streaking (Figure 4B). With this evidence, it was concluded that MLKL was not dissociating from the fibers during the SDD-AGE process and that the MLKL-containing fibers seen in Figure 1 were indeed distinct from the RIPK1/RIPK3 fibers. Furthermore, when RIPK3 was immuno-depleted from the cell lysates, the MLKL fibers remained intact4, confirming that MLKL fibers and RIPK1/RIPK3 fibers are indeed distinct entities.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57498/57498fig1.jpg" /> 
Figure 1. Examination of amyloid-like fibers during necroptosis. Whole cell lysates were harvested from cells undergoing TNF-induced necroptosis and subjected to SDD-AGE. Western blots were performed for RIPK1, RIPK3, and MLKL. The MLKL-containing fibers exhibited a distinct migration pattern from the RIPK1/RIPK3-containing fibers. The MLKL monomer is barely visible under this condition. <a href="/files/ftp_upload/57498/57498fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57498/57498fig2.jpg" /> 
Figure 2. Experimental protocol. (A) Schematic of the two-dimensional semi-denaturing detergent agarose gel electrophoresis (2D SDD-AGE). Begin by loading the sample in the right most lane, labeled in red. After termination of the first dimension run, rotate the gel 90&#176; counter-clockwise. Run the second dimension electrophoresis. Solid arrow indicates the direction of the first dimension run, dotted arrow indicates the direction of the second dimension run. (B) Schema of transfer by capillary action. <a href="/files/ftp_upload/57498/57498fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57498/57498fig3.jpg" /> 
Figure 3. Possible results. (A) The expected migration pattern of amyloid or amyloid-like fibers after the first dimension of SDD-AGE. Solid arrow indicates the direction of migration. (B) The expected migration pattern of the SDD-AGE-stable (no degradation or dissociation) amyloid or amyloid-like fibers after 2D SDD-AGE. Solid arrow indicates the direction of the first dimension SDD-AGE. Dotted arrow indicates the direction of the second dimension SDD-AGE. (C) The expected migration pattern of the non-SDD-AGE-stable amyloid or amyloid-like fibers. Solid arrow indicates the direction of the first dimension SDD-AGE. Dotted arrow indicates the direction of the second dimension SDD-AGE. <a href="/files/ftp_upload/57498/57498fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57498/57498fig4.jpg" /> 
Figure 4. Migration of MLKL-containing fibers during 1D and 2D SDD-AGE. (A) Whole cell lysates were harvested from cells stimulated with TSZ to undergo necroptosis. The lysates were subjected to SDD-AGE, and a Western blot was performed for MLKL. A characteristic smear was observed. (B) Whole cell lysates were harvested from cells stimulated with TSZ to undergo necroptosis. The lysates were subjected to 2D SDD-AGE and a Western blot was performed for MLKL. A sharp line at a 45&#176; angle was observed, indicating that the fibers do not undergo dissociation during SDD-AGE. <a href="/files/ftp_upload/57498/57498fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

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Nanyang Technological University

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

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 &#176;C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

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.

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 ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...