1Whitehead Institute for Biomedical Research, 2Department of Biology, MIT - Massachusetts Institute of Technology, 3Howard Hughes Medical Institute
Halfmann, R., Lindquist, S. Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis. J. Vis. Exp. (17), e838, doi:10.3791/838 (2008).
Part 1: Preparing the gel
Part 2: Preparing samples
Part 3: Transfer
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)
100 mM Tris 7.5
50 mM NaCl
10 mM BME (add fresh)
protease inhibitors (add fresh)
4X Sample Buffer
bromophenol blue to preference
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°C can be used, which will restore most amyloids to monomeric protein.
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.).
|zymolyase 100T||Seikagaku Corporation||120493-1|
|Halt Protease Inhibitor Cocktail||Thermo Fisher Scientific, Inc.||78429|
|Hybond-C extra nitrocellulose||Amersham||RPN303E|
|GB004 blotting paper||Whatman, GE Healthcare||10427926|
|GB002 (3MM Chr) blotting paper||Whatman, GE Healthcare||3030-917|
1. Kryndushkin, D.S., Alexandrov, I.M., Ter-Avanesyan, M.D. & Kushnirov, V.V. Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104. J. Biol. Chem. 278, 49636-49643 (2003).
2. Alexandrov, I.M., Vishnevskaya, A.B., Ter-Avanesyan, M.D. & Kushnirov, V.V. Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION. J. Biol. Chem. 283, 15185-15192 (2008).
3. Allen, K.D. et al. Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+]. Genetics 169, 1227-1242 (2005).
4. Aron, R., Higurashi, T., Sahi, C. & Craig, E.A. J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation. The EMBO journal 26, 3794-3803 (2007).
5. Bagriantsev, S.N., Kushnirov, V.V. & Liebman, S.W. Analysis of amyloid aggregates using agarose gel electrophoresis. Methods in Enzymology 412, 33-48 (2006).
6. Borchsenius, A.S., Muller, S., Newnam, G.P., Inge-Vechtomov, S.G. & Chernoff, Y.O. Prion variant maintained only at high levels of the Hsp104 disaggregase. Current Genetics 49, 21-29 (2006).
7. Salnikova, A.B., Kryndushkin, D.S., Smirnov, V.N., Kushnirov, V.V. & Ter-Avanesyan, M.D. Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids. J. Biol. Chem. 280, 8808-8812 (2005).
8. Tank, E.M., Harris, D.A., Desai, A.A. & True, H.L. Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability. Mol. Cell. Biol. 27, 5445-5455 (2007).
9. Douglas, P.M. et al. Chaperone-dependent amyloid assembly protects cells from prion toxicity. Proc. Natl. Acad. Sci. USA 105, 7206-7211 (2008).
10. Nagy, B., Costello, R., and Csako, G. Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping. Analytical Biochemistry. 231, 40-45 (1995).