SDD-AGE

In biochemistry and molecular biology, SDD-AGE is short for Semi-Denaturating Detergent Agarose Gel Electrophoresis. This is a method for detecting and characterizing large protein polymers which are stable in 2% SDS at room temperature, unlike most large protein complexes. This method is very useful for studying prions and amyloids, which are characterized by the formation of proteinaceous polymers.[1][2][3][4][5][6] Agarose is used for the gel since the SDS-resistant polymers are large (in the 200-4000+ kDa range) and cannot enter a conventional polyacrylamide gel, which has small pores. Agarose on the other hand has large pores, which allows for the separation of polymers.

Example of a resulting western blott after SDD-AGE electrophoretic separation, staining by specific antibodies

Use of this method allowed researchers to understand that at least some types of prion aggregates existed in a two-level structure - protein molecules grouped into polymers, which are very stable and withstand treatment with 2% SDS at room temperature, and aggregates, which are bundles of polymers, that dissociate under these conditions.

Differences in the size of polymers can indicate the efficiency of polymer fragmentation in vivo.

History

The method was created in the Molecular Genetics laboratory of the Russian Cardiology Research Institute and was published in 2003 by Kryndushkin et al.[1] The original method used a TAE buffering system and incorporated a modified vacuum blotting system for the transfer of proteins onto a membrane (originally PVDF). The modified vacuum blotting system is actually a vacuum-assisted capillary transfer, since the vacuum only helps fluid that has already gone through the gel and membrane to leave the system.

Variations

Other modifications have also been used, such as the one described in Bagriantsev et al.,[7] using traditional wet transfer and a TGB buffering system, and others using semi-dry transfer or capillary transfer.[8]

DD-AGE, a further variation of the method that uses fully denaturing conditions - including reducing agents such as dithiothreitol (DTT) and heat denaturation at 95°C - is suitable for the analysis of heat-stable inclusion bodies of polyglutamine proteins.[9]

References
  1. Kryndushkin DS; Alexandrov IM; Ter-Avanesyan MD; Kushnirov VV (2003). "Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104". Journal of Biological Chemistry. 278 (49): 49636–43. doi:10.1074/jbc.M307996200. PMID 14507919.
  2. Salnikova AB; Kryndushkin DS; Smirnov VN; Kushnirov VV; Ter-Avanesyan MD (2005). "Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids". Journal of Biological Chemistry. 280 (10): 8808–12. doi:10.1074/jbc.M410150200. PMID 15618222.
  3. Meriin AB; Zhang X; Alexandrov IM; Salnikova AB; Ter-Avanesian MD; Chernoff YO; Sherman MY (2007). "Endocytosis machinery is involved in aggregation of proteins with expanded polyglutamine domains". FASEB Journal. 21 (8): 1915–25. doi:10.1096/fj.06-6878com. PMID 17341688. S2CID 24922939.
  4. Shkundina IS; Kushnirov VV; Tuite MF; Ter-Avanesyan MD (2006). "The role of the N-terminal oligopeptide repeats of the yeast Sup35 prion protein in propagation and transmission of prion variants". Genetics. 172 (2): 827–35. doi:10.1534/genetics.105.048660. PMC 1456247. PMID 16272413.
  5. Alexandrov IM; Vishnevskaya AB; Ter-Avanesyan MD; Kushnirov VV (2008). "Appearance and propagation of polyglutamine-based amyloids in yeast: tyrosine residues enable polymer fragmentation". Journal of Biological Chemistry. 283 (22): 15185–92. doi:10.1074/jbc.M802071200. PMC 2397454. PMID 18381282.
  6. Alberti S; Halfmann R; King O; Kapila A; Lindquist S (2009). "A systematic survey identifies prions and illuminates sequence features of prionogenic proteins". Cell. 137 (1): 146–58. doi:10.1016/j.cell.2009.02.044. PMC 2683788. PMID 19345193.
  7. Bagriantsev SN; Kushnirov VV; Liebman SW (2006). Analysis of amyloid aggregates using agarose gel electrophoresis. Methods in Enzymology. Vol. 412. pp. 33–48. doi:10.1016/S0076-6879(06)12003-0. ISBN 9780121828172. PMID 17046650.
  8. Halfmann R; Lindquist S (2008). "Screening for amyloid aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis". Journal of Visualized Experiments (17). doi:10.3791/838. PMC 2723713. PMID 19066511.
  9. Weber JJ; Golla M; Guaitoli G; Wanichawan P; Hayer SN; Hauser S; Krahl AC; Nagel M; Samer S; Aronica E; Carlson CR; Schöls L; Riess O; Gloeckner CJ; Nguyen HP; Hübener-Schmid J (2017). "A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3". Brain. 140 (5): 1280–1299. doi:10.1093/brain/awx039. PMID 28334907.
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