biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...

and

molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...

, SDD-AGE is short for Semi-Denaturating Detergent Agarose Gel Electrophoresis. This is a method for detecting and characterizing large

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

polymer A polymer (; Greek '' poly-'', "many" + ''-mer'', "part") is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...

s which are stable in 2% SDS at

room temperature Colloquially, "room temperature" is a range of air temperatures that most people prefer for indoor settings. It feels comfortable to a person when they are wearing typical indoor clothing. Human comfort can extend beyond this range depending on ...

, unlike most large

protein complex A protein complex or multiprotein complex is a group of two or more associated polypeptide chains. Protein complexes are distinct from multienzyme complexes, in which multiple catalytic domains are found in a single polypeptide chain. Protein c ...

es. This method is very useful for studying

prion Prions are misfolded proteins that have the ability to transmit their misfolded shape onto normal variants of the same protein. They characterize several fatal and transmissible neurodegenerative diseases in humans and many other animals. It ...

s and

amyloid Amyloids are aggregates of proteins characterised by a Fibril, fibrillar morphology of 7–13 Nanometer, nm in diameter, a beta sheet (β-sheet) Secondary structure of proteins, secondary structure (known as cross-β) and ability to be Staining, ...

s, which are characterized by the formation of

proteinaceous Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

polymers.

Agarose Agarose is a heteropolysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is o ...

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 Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their ...

, 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 Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respond ...

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 Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

''.

History

The method was created in the

Molecular Genetics Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the ...

laboratory of the Russian Cardiology Research Institute and was published in 2003 by Kryndushkin et al. The original method used a

TAE buffer TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA. In molecular biology it is used in agarose electrophoresis typically for the separation of nucleic acids such as DNA and RNA. It is made up of Tris-acetate ...

ing system and incorporated a modified vacuum blotting system for the transfer of proteins onto a membrane (originally

PVDF Polyvinylidene fluoride or polyvinylidene difluoride (PVDF) is a highly non-reactive thermoplastic fluoropolymer produced by the polymerization of vinylidene difluoride. PVDF is a specialty plastic used in applications requiring the highest pur ...

). The modified vacuum blotting system is actually a vacuum-assisted capillary transfer, since the

vacuum A vacuum is a space devoid of matter. The word is derived from the Latin adjective ''vacuus'' for "vacant" or "void". An approximation to such vacuum is a region with a gaseous pressure much less than atmospheric pressure. Physicists often dis ...

only helps fluid that has already gone through the

gel A gel is a semi-solid that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still dif ...

and membrane to leave the system.

Variations

Other modifications have also been used, such as the one described in Bagriantsev et al., using traditional wet transfer and a TGB buffering system, and others using semi-dry transfer or capillary transfer. DD-AGE, a further variation of the method that uses fully denaturing conditions - including reducing agents such as

dithiothreitol Dithiothreitol (DTT) is the common name for a small-molecule redox reagent also known as Cleland's reagent, after W. Wallace Cleland. DTT's formula is C4H10O2S2 and the chemical structure of one of its enantiomers in its reduced form is shown on ...

(DTT) and heat denaturation at 95°C - is suitable for the analysis of heat-stable inclusion bodies of polyglutamine proteins.

References

{{DEFAULTSORT:Sdd-Age Protein methods Molecular biology