Name: Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (Video) | JoVE Journal
Uploaded: 2008-07-16T17:18:00.000+00:00
Duration: 10 min 3 s
Description: Learn SDD-AGE for screening amyloid-like polymers and prions in protein analysis. This scalable method uses standard electrophoresis equipment to separate SDS-resistant aggregates for neuroscience and biochemistry research.

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5 minutes

We thank Simon Alberti for assistance with developing this protocol. This work was supported by a grant from the National Institutes of Health (GM25874), a Howard Hughes Medical Institute Investigatorship (to S.L.), and a National Science Foundation predoctoral training grant (to R.H.).

Part 1: Preparing the gel
<ol>
<li>Assemble the gel casting tray. Standard equipment for horizontal DNA electrophoresis can be used. For large numbers of samples, we prepare a 20 cm x 24 cm slab with up to four 50-well combs. Make sure the slab does not have scratches, as these can distort the blot image.</li>
<li>Create a 1.5% agarose solution (medium or high gel-strength, low EEO) in 1X TAE. The volume should be enough to completely submerge the comb teeth -- you will want to load as much sample as possible to maximize detection. Microwave the mixture until the agarose is completely dissolved.</li>
<li>Rapidly add SDS to 0.1% from a 10% stock. Swirl to mix. If some agarose solidifies during this step, redissolve using a hot plate and be careful to avoid boiling.</li>
<li>Pour the solution into the casting tray. Use a comb to rake out any bubbles, as they could later interfere with transfer.</li>
<li>After gel has set, remove combs and place the gel into the gel tank. Completely submerge the gel in 1X TAE containing 0.1% SDS.</li>
</ol>
Part 2: Preparing samples
<ol>
<li>For high-throughput analysis of yeast lysates, we start with 2-ml cultures grown overnight with rapid agitation in 96-well blocks. In this case, each culture is overexpressing a protein of interest. When analyzing low abundance proteins, larger culture volumes must be used </li>
<li>Harvest cells by centrifugation at 2000 RCF for 5 min at room temperature. </li>
<li>Resuspend cells in water and centrifuge again.</li>
<li>Resuspend in 1 ml spheroplasting solution. Incubate for approximately 30 min at 30&deg;C (you may confirm spheroplasting efficiency by microscopy). </li>
<li>Centrifuge at 800 RCF for 5 min at room temperature. Completely remove supernatant.</li>
<li>Resuspend pelleted spheroplasts in 100 ml lysis buffer. </li>
<li>Cover the block with tape and vortex on high speed for 2 min.</li>
<li>Pellet cellular debris at 4000 RCF for 2 min. </li>
<li>Carefully remove supernatant to a new container, e.g., a 96-well PCR plate. </li>
<li>If desired, determine the protein concentration of the lysates.</li>
<li>Add 4X sample buffer to the samples to generate lysates containing 1X sample buffer. Incubate for 5 min at room temperature.</li>
<li>Load gel. If desired, save half of the sample volume and boil it for SDS-PAGE analysis. In order to monitor the extent of transfer later on, include a pre-stained SDS-PAGE marker. Additionally, a molecular weight marker consisting of very large proteins (e.g., chicken pectoralis extract) can be used to estimate sizes of the resolved complexes.</li>
<li>Run at low voltage (&le; 3 V/cm gel length) until the dye front reaches ~1 cm from the end of the gel. This will take several hours. It is important that the gel remain cool; otherwise diffusion of low molecular weight protein (e.g., monomers) can limit their detection. </li>
</ol>
Part 3: Transfer
<ol>
<li>Cut a piece of nitrocellulose to the same dimensions as the gel. </li>
<li>Cut 20 pieces of GB004 and 8 pieces of GB002 blotting paper, to the same dimensions as the gel. Cut an additional piece of GB004 to be used as a wick; make it about 20 centimeters wider than the gel. </li>
<li>Immerse the nitrocellulose, wick, and 4 pieces of GB002 in 1X TBS. </li>
<li>In a plastic container, assemble a stack of papers as follows: 20 pieces of dry GB004, then 4 pieces of dry GB002, then one piece of pre-wet GB002. Lay the nitrocellulose on top of this stack.</li>
<li>Rinse the gel on the casting tray briefly in water to remove excess running buffer. Then, carefully begin to slide the gel off the tray onto the stack. While sliding the gel off the tray, douse the membrane with TBS as necessary. The extra buffer helps prevent bubbles from becoming trapped under the gel. A transfer pipette works well for this purpose.</li>
<li>After the gel has been moved to the stack, check thoroughly for bubbles. If any are present, lift the edge of the gel and reapply buffer until the bubbles can be worked out.</li>
<li>Put the remaining three pre-wetted GB002 pieces on top of the gel. Ensure thorough contact between all layers by rolling a pipette firmly across the top of the stack.</li>
<li>Flank the transfer stack with two elevated trays containing TBS. Drape the pre-wet wick across the stack such that either end of the wick is submerged in TBS.</li>
<li>Cover the assembled transfer stack with an additional plastic tray bearing extra weight (e.g., a 500 ml bottle of water).</li>
<li>Allow the transfer to proceed for a minimum of three hours, or overnight.</li>
<li>After transfer, the membrane can be processed by standard Western blotting.</li>
</ol>
Spheroplasting Solution 1.2 M D-sorbitol 0.5 mM MgCl2 20 mM Tris, pH 7.5 50 mM BME (add fresh) 0.5 mg/ml Zymolyase 100T (add fresh)
Lysis Buffer 100 mM Tris 7.5 50 mM NaCl 10 mM BME (add fresh) protease inhibitors (add fresh) 4X Sample Buffer 2X TAE 20% glycerol 8% SDS bromophenol blue to preference

<ol><li>Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yeast+%5BPSI%2B%5D+prion+aggregates+are+formed+by+small+Sup35+polymers+fragmented+by+Hsp104%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104.</a> J. Biol. Chem. 278, 49636-49643 (2003).</li>
<li>Alexandrov, I. M., Vishnevskaya, A. B., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Appearance+and+Propagation+of+Polyglutamine-based+Amyloids+in+Yeast%3A+TYROSINE+RESIDUES+ENABLE+POLYMER+FRAGMENTATION%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION.</a> J. Biol. Chem. 283, 15185-15192 (2008).</li>
<li>Allen, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hsp70+chaperones+as+modulators+of+prion+life+cycle%3A+novel+effects+of+Ssa+and+Ssb+on+the+Saccharomyces+cerevisiae+prion+%5BPSI%2B%5D%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].</a> Genetics. 169, 1227-1242 (2005).</li>
<li>Aron, R., Higurashi, T., Sahi, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((J-protein+co-chaperone+Sis1+required+for+generation+of+%5BRNQ%2B%5D+seeds+necessary+for+prion+propagation.%5BTitle%5D)+AND+%22The+EMBO+journal%22%5BJournal%5D)">J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation.</a> The EMBO journal. 26, 3794-3803 (2007).</li>
<li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+amyloid+aggregates+using+agarose+gel+electrophoresis%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Analysis of amyloid aggregates using agarose gel electrophoresis.</a> Methods in Enzymology. 412, 33-48 (2006).</li>
<li>Borchsenius, A. S., Muller, S., Newnam, G. P., Inge-Vechtomov, S. G., Chernoff, Y. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prion+variant+maintained+only+at+high+levels+of+the+Hsp104+disaggregase%5BTitle%5D)+AND+%22Current+Genetics%22%5BJournal%5D)">Prion variant maintained only at high levels of the Hsp104 disaggregase.</a> Current Genetics. 49, 21-29 (2006).</li>
<li>Salnikova, A. B., Kryndushkin, D. S., Smirnov, V. N., Kushnirov, V. V., Ter-Avanesyan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonsense+suppression+in+yeast+cells+overproducing+Sup35+%28eRF3%29+is+caused+by+its+non-heritable+amyloids%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids.</a> J. Biol. Chem. 280, 8808-8812 (2005).</li>
<li>Tank, E. M., Harris, D. A., Desai, A. A., True, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prion+protein+repeat+expansion+results+in+increased+aggregation+and+reveals+phenotypic+variability%5BTitle%5D)+AND+%22Mol.+Cell.+Biol%22%5BJournal%5D)">Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.</a> Mol. Cell. Biol. 27, 5445-5455 (2007).</li>
<li>Douglas, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chaperone-dependent+amyloid+assembly+protects+cells+from+prion+toxicity%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Chaperone-dependent amyloid assembly protects cells from prion toxicity.</a> Proc. Natl. Acad. Sci. USA. 105, 7206-7211 (2008).</li>
<li>Nagy, B., Costello, R., Csako, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Downward+blotting+of+proteins+in+a+model+based+on+apolipoprotein%28a%29+phenotyping%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping.</a> Analytical Biochemistry. 231, 40-45 (1995).</li>
</ol>

SDD-AGE was first reported by Kryndushkin et al.1, to study SDS-resistant complexes of the [PSI+] prion in yeast, and has since found widespread use studying both prion and non-prion aggregates 2-9. However, transfer of the proteins to a membrane following electrophoresis in an agarose gel is problematic, and can result in a distorted blot image 5. Additionally, the submerged electroblotting technique most commonly used introduces practical limitations for the size of the gel and thus the number of samples that can be processed. We have addressed these problems by employing downward capillary transfer 10, a simple procedure which uses a stack of dry blotting papers to transfer proteins from the gel to a nitrocellulose membrane. Capillary transfer prevents distortion and allows large gels to be processed easily. There are a few things to consider before using SDD-AGE. For crude samples (e.g., lysates), immunodetection of specific proteins is necessary. SDD-AGE does not fully denature the protein complexes of interest, so the protein(s) to be detected must bear an epitope tag outside of the amyloidogenic region. Lysates can generally be prepared as they would be for a normal SDS-PAGE, with two important differences. First, increased care must be taken to prevent degradation by proteolysis. The partially denaturing conditions used here are not sufficient to inactivate proteases, and can also make target proteins more susceptible to proteolysis. Use a complete protease inhibitor cocktail at at least two-fold the recommended concentration. Second, heating the samples should be avoided. If an all-monomer negative control is desired, for instance to confirm that high-molecular-weight species are not due to covalent modifications, a 10-minute incubation at 95&deg;C can be used, which will restore most amyloids to monomeric protein.

<table><tbody><tr><td>zymolyase 100T</td><td>Seikagaku Corporation</td><td>120493-1</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>Thermo Fisher Scientific, Inc.</td><td>78429</td><td></td></tr><tr><td>Hybond-C extra nitrocellulose</td><td>Amersham</td><td>RPN303E</td><td></td></tr><tr><td>GB004 blotting paper</td><td>Whatman, GE Healthcare</td><td>10427926</td><td></td></tr><tr><td>GB002 (3MM Chr) blotting paper</td><td>Whatman, GE Healthcare</td><td>3030-917</td><td></td></tr><tr><td>Agarose (UltraPure)</td><td>Invitrogen</td><td>15510-027</td><td></td></tr></tbody></table>

screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis

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 ...

Hello, I'm Randall Hoffman, a graduate student in Susan Linus Laboratory in the biology department at MIT. Today I'm going to introduce you to semi denaturing detergent agro rose, gel electrophoresis, or SDD age. Our lab uses this technique to screen for prions and other alogenic proteins.Our version of SDD age is simple and reliable and can be scaled to accommodate any number of samples. Amyloids are exceptionally resistant to denaturation by SDS, which allows them to be separated from other proteins using SDDH. However, data from SDDH cannot by itself be used to confirm that something is amyloid.To do that, you need to use other techniques. With that in mind, let me show you. S-D-D-H-S-D-D-H can be performed using standard equipment for horizontal DNA electrophoresis.First, you need to assemble the gel casting tray. The size of the apparatus will depend on how many samples you have. For a large number of samples, we use a 20 by 24 centimeter slab, and up to four 50 well combes.To prepare the gel, you'll need medium or high gel strength low, EEO agros, one XTAE buffer, and a 10%stock solution of SDS Make a 1.5%AROS solution and one XTAE. You'll want to make the gel as thick as possible so you can load the most sample and maximize detection. Heat the mixture in the microwave until the aros is completely dissolved.Quickly add the 100 XSDS stock to 0.1%and swirl to mix. If some agro solidifies during this step, red, dissolve it on a hot plate. Be careful not to let it boil.After adding SDS as the solution will easily bubble over, pour this solution into the casting tray while it is still hot. Then use a comb to get rid of any bubbles which may interfere later on during gel transfer. After the gel is completely cooled and solidified, carefully remove the combs and place the gel into the tank.Completely submerge the gel in one XTAE with 0.1%SDS. You are now ready to load your samples. The protein samples used in SDDH are prepared just like in an SDS page with two important exceptions.First, you should avoid heating the samples which could denature the aggregates into protein monomers. Second, take care to avoid protein degradation. Because the denaturing conditions used here are not sufficient to inactivate all proteases, we recommend using at least twice the recommended concentration of a complete protease inhibitor cocktail.In this demonstration, we'll use lysates from yeast cells that are over expressing a particular protein of interest. We generally use two mil overnight. Cultures grown in 96, well blocks available from VWR, but when analyzing low abundance proteins, larger culture volumes must be used.Harvest the cells by centrifuging the culture at 2000 RCF for five minutes at room temperature following centrifugation, remove the supra natant and wash the cells by Resus suspending in water and centrifuging again after the second spin, remove the supernatant and resin the cells in spare plating solution. The zy in the solution digest the cell walls, which will allow you to ly the cells efficiently and a small volume of lysis buffer. Incubate for 30 minutes at 30 degrees Celsius.Pellet the spare plastic cells this time at 800 RCF for five minutes. Remove the sate completely and resuspend each pellet and 100 microliters of lysis buffer. Next, cover the block with tape and vortex on high speed for two minutes.This helps all the sphere plastics to LY P out the cellular debris at 4, 000 RCF. For two minutes. Carefully transfer the S natin to a 96 well PCR plate.Typically, we analyze hundreds of samples at a time in our laboratory, so to assist us with this is a liquid handling system. If desired, determine the protein concentration of the lysates. Next, add four x sample buffer to the samples to generate lysates containing one x sample buffer incubate for five minutes at room temperature.Now load the samples into the gel. If desired, save half of the samples for SDS page analysis. Also, load molecular weight markers, including prestained markers and a very high molecular weight marker such as chicken flector s extract.If desired, it is important to keep the gel cool to prevent diffusion of low molecular weight proteins, so you may want to run it in the cold room. Run the gel at low voltage less than three volts per centimeter of the gel length. Run the gel for several hours until the diare reaches about one centimeter from the end of the gel.Because amyloid is not denatured by 2%SDS at room temperature, it will remain intact while other complexes, including most non amyloid aggregates, dissociate in the presence of 2%SDS. Thus, most proteins will be monomeric and run quickly through the gel. While the SDS resistant high molecular weight amyloids will migrate more slowly.After this, the gel will be ready for transfer. Although multiple transfer methods can be used, we obtain the best results using capillary transfer. Since capillary transfer can accommodate any size of gel, it is practical to analyze hundreds of samples in capillary transfer.A stack of dry blotting papers acts to draw buffer through the gel depositing proteins onto a nitrocellulose membrane placed between the papers and the gel. You'll need to cut a piece of nitrocellulose, such as high bond C, extra to the same dimensions as the gel. You'll also need to cut 20 pieces of GBO oh four and eight pieces of GBO oh two blotting paper to the same size as the gel.GB oh four is very thick and absorbent. While GBO oh two is much thinner and has a finer texture, which ensures more even contact with the membrane. Cut an additional piece of GBO oh four to be used as a wick and make it about 20 centimeters wider than the gel immersed.The nitrous UL WIC and four pieces of GBO oh two and one XTBS in a plastic container assemble a stack of papers as follows, 20 pieces of dry GBO oh four. Then four pieces of dry GBO oh two. Then one piece of pre wetted GBO oh two.Lay the nitrocellulose on top of this stack. Rinse the gel on the casting tray briefly in water to remove excess running buffer. Then carefully begin to slide the gel off the tray onto the stack while sliding the gel off the tray dows the membrane with TBS as necessary.The extra buffer helps prevent bubbles from becoming trapped into the gel. The transfer pipette works well for this purpose after the gel has been completely moved to the stack. Check thoroughly for bubbles, if any are present.Lift the edge of the gel and reapply buffer until the bubbles can be worked out. Put the remaining three pre wetted GBO oh two pieces on top of the gel. Ensure thorough contact between all layers by rolling a pipette firmly across the top of the stack.Flank the transfer stack with two elevated trays containing TBS drape the pre-wet wick across the stack, such that either end of the wick is submerged. In TBS cover the assembled transfer stack with an additional plastic tray bearing extra weight such as a 500 mil bottle of water. The wick will draw a buffer from the trays and the dry blotting papers will draw the buffer through the gel over the course of several hours.This will deposit the proteins onto the membrane, allow the transfer to proceed for a minimum of three hours or overnight. After the capillary transfer is complete, the membrane can be processed by standard western blotting. Here you see a typical blot of proteins that were over expressed in a yeast strain that predisposes certain proteins to form amyloid.You can clearly see that some of our candidates form amyloid or high molecular weight SDS resistant species very well like those shown here and here. Others, however, do not form aggregates and are completely monomeric such as these and these. Additionally, you can see that the size of the amyloid polymers differs depending on the protein, which you can see in these two lanes.We've just showed you a simple method for analyzing high molecular weight, insoluble aggregates, such as amyloids of candidate prions. We hope you've enjoyed watching our presentation and find this technique useful and studies of Amy immunogenic proteins. Thanks for watching and good luck with your research.

screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

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Learn SDD-AGE for screening amyloid-like polymers and prions in protein analysis. This scalable method uses standard electrophoresis equipment to separate SDS-resistant aggregates for neuroscience and biochemistry research.

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

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