View source for SDD-AGE

{{Short description|Method for detecting large protein polymers}}
[[File:SDD-AGE (small and large polymers).jpg|thumb|upright|alt=Example of a resulting western blott after SDD-AGE electrophoretic separation, staining by specific antibodies]]
In [[biochemistry]] and [[molecular biology]], '''SDD-AGE''' is short for '''S'''emi-'''D'''enaturating '''D'''etergent '''A'''garose '''G'''el '''E'''lectrophoresis. This is a method for detecting and characterizing large [[protein]] [[polymer]]s which are stable in 2% [[Sodium dodecyl sulfate|SDS]] at [[room temperature]], unlike most large [[protein complex]]es. This method is very useful for studying [[prion]]s and [[amyloid]]s, which are characterized by the formation of [[proteinaceous]] polymers.<ref name="kryndushkin"/><ref>{{Cite journal|author1=Salnikova AB |author2=Kryndushkin DS |author3=Smirnov VN |author4=Kushnirov VV |author5=Ter-Avanesyan MD | title= Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids|journal= Journal of Biological Chemistry|volume= 280|pages= 8808–12  |year=2005 |doi= 10.1074/jbc.M410150200|issue= 10|pmid= 15618222|doi-access= free}}</ref><ref>{{Cite journal|author1=Meriin AB |author2=Zhang X |author3=Alexandrov IM |author4=Salnikova AB |author5=Ter-Avanesian MD |author6=Chernoff YO |author7=Sherman MY | title= Endocytosis machinery is involved in aggregation of proteins with expanded polyglutamine domains|journal= FASEB Journal|volume= 21|pages= 1915–25  |year=2007 |doi= 10.1096/fj.06-6878com|pmid=17341688|issue= 8|doi-access=free |s2cid=24922939 }}</ref><ref>{{Cite journal|author1=Shkundina IS |author2=Kushnirov VV |author3=Tuite MF |author4=Ter-Avanesyan MD | title= The role of the N-terminal oligopeptide repeats of the yeast Sup35 prion protein in propagation and transmission of prion variants  |journal= Genetics|volume= 172|pages= 827–35  |year=2006 |doi= 10.1534/genetics.105.048660|issue= 2|pmid= 16272413|pmc= 1456247}}</ref><ref>{{Cite journal|author1=Alexandrov IM |author2=Vishnevskaya AB |author3=Ter-Avanesyan MD |author4=Kushnirov VV | title= Appearance and propagation of polyglutamine-based amyloids in yeast: tyrosine residues enable polymer fragmentation|journal= Journal of Biological Chemistry|volume= 283|pages= 15185–92  |year=2008 |doi= 10.1074/jbc.M802071200|issue= 22|pmid= 18381282|pmc= 2397454|doi-access=free }}</ref><ref>{{Cite journal|author1=Alberti S |author2=Halfmann R |author3=King O |author4=Kapila A |author5=Lindquist S | title=A systematic survey identifies prions and illuminates sequence features of prionogenic proteins|journal= Cell|volume= 137|pages= 146–58|year=2009 |doi=10.1016/j.cell.2009.02.044|pmid=19345193|issue= 1|pmc= 2683788}}</ref> [[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.
Use of this method allowed researchers to understand that at least some types of prion aggregates existed in a two-level structure - [[protein molecule]]s 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|Molecular Genetics]] laboratory of the Russian Cardiology Research Institute and was published in 2003 by Kryndushkin et al.<ref name="kryndushkin">{{Cite journal|author1=Kryndushkin DS |author2=Alexandrov IM |author3=Ter-Avanesyan MD |author4=Kushnirov VV | title= Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104  |journal= Journal of Biological Chemistry|volume= 278|pages= 49636–43  |year=2003 |doi= 10.1074/jbc.M307996200|issue= 49|pmid= 14507919|doi-access= free}}</ref> The original method used a [[TAE buffer]]ing system and incorporated a modified vacuum blotting system for the transfer of proteins onto a membrane (originally [[Polyvinylidene fluoride|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.,<ref>{{Cite book|author1=Bagriantsev SN |author2=Kushnirov VV |author3=Liebman SW |chapter=Analysis of Amyloid Aggregates Using Agarose Gel Electrophoresis | title= Amyloid, Prions, and Other Protein Aggregates, Part B  |series= Methods in Enzymology|volume= 412|pages= 33–48  |year=2006 |doi=  10.1016/S0076-6879(06)12003-0 |pmid=17046650|isbn=9780121828172 }}</ref> using traditional wet transfer and a TGB buffering system, and others using semi-dry transfer or capillary transfer.<ref>{{Cite journal|author1=Halfmann R |author2=Lindquist S | title= Screening for amyloid aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis |journal= Journal of Visualized Experiments |year=2008 |doi=  10.3791/838|issue= 17|pmc=2723713 |pmid=19066511}}</ref>
DD-AGE, a further variation of the method that uses fully [[Denaturation (biochemistry)|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 disease|polyglutamine proteins]].<ref>{{Cite journal|author1=Weber JJ |author2=Golla M |author3=Guaitoli G |author4=Wanichawan P |author5=Hayer SN |author6=Hauser S |author7=Krahl AC |author8=Nagel M |author9=Samer S |author10=Aronica E |author11=Carlson CR |author12=Schöls L |author13=Riess O |author14=Gloeckner CJ |author15=Nguyen HP |author16=Hübener-Schmid J |title = A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3 |journal= Brain |volume=140 |pages= 1280–1299 |year= 2017 |issue=5 |doi= 10.1093/brain/awx039 |pmid = 28334907|doi-access=free }}</ref>
==References==
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{{Reflist}}
{{DEFAULTSORT:Sdd-Age}}
[[Category:Protein methods]]
[[Category:Molecular biology]]