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, a ...

and

molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...

, 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 residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

polymer A polymer () is a chemical substance, substance or material that consists of very large molecules, or macromolecules, that are constituted by many repeat unit, repeating subunits derived from one or more species of monomers. Due to their br ...

s which are stable in 2% SDS at

room temperature Room temperature, colloquially, denotes the range of air temperatures most people find comfortable indoors while dressed in typical clothing. Comfortable temperatures can be extended beyond this range depending on humidity, air circulation, and ...

, 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 multidomain enzymes, in which multiple active site, catalytic domains are found in a single polypeptide chain. ...

es. This method is very useful for studying

prion A prion () is a Proteinopathy, misfolded protein that induces misfolding in normal variants of the same protein, leading to cellular death. Prions are responsible for prion diseases, known as transmissible spongiform encephalopathy (TSEs), w ...

s and

amyloid Amyloids are aggregates of proteins characterised by a fibrillar morphology of typically 7–13 nm in diameter, a β-sheet secondary structure (known as cross-β) and ability to be stained by particular dyes, such as Congo red. In the human ...

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, respon ...

polymers.

Agarose Agarose is a heteropolysaccharide, generally extracted from certain red algae. 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 on ...

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 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, respon ...

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, an ...

''.

History

The method was created in the

Molecular Genetics Molecular genetics is a branch 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 st ...

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. Its chemical formula is (C2H2F2)''n''. PVDF is a specialty plastic used ...

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

vacuum A vacuum (: vacuums or vacua) is space devoid of matter. The word is derived from the Latin adjective (neuter ) meaning "vacant" or "void". An approximation to such vacuum is a region with a gaseous pressure much less than atmospheric pressur ...

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., 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 an organosulfur compound with the formula . A colorless compound, it is classified as a dithiol and a diol. DTT is redox reagent also known as Cleland's reagent, after W. Wallace Cleland. The reagent is commonly used in ...

(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