Immobilized disulfide reductants

The use of immobilized disulfide reductants thus has the following advantages over solution phase agents ... 

Immobilized disulfide reductants can be used to reduce all types of biological disulfides without liberating product or by-product contaminants. 

Soluble components that interfere with the assay of free thiol groups are not present if immobilized disulfide reductants are used. 

Immobilized disulfide reductants easily can be regenerated and reused many times. 

Immobilized dihydrolipoamide (thioctic acid) (Gorecki and Patchornick, 1973 Gorecki and Patchornick, 1975) and immobilized N-acetyl-homocysteine thiolactone (Eldjarn and Jellum, 1963 Jellum, 1964) are the two most commonly used immobilized disulfide reductants. In addition, immobilized TCEP provides a reducing matrix that is free of thiols (Thermo Fisher). Such immobilized reductants successfully can be used to reduce many types of biological disulfides, including small molecules like oxidized glutathione and bovine insulin. They... 

Immobilized disulfide reductants may be synthesized as described in Hermanson et al. (1992) or obtained commercially (Thermo Fisher). 

Small molecules containing disulfide bonds (such as cystine-containing peptides) may be reduced and isolated simply by removing the immobilized reductant. Separation of reduced molecules from reductant is much more difficult if a soluble reducing agent is used with low-molecular-weight disulfides. 

Apply to the column 1.0ml of protein solution (dissolved in equilibration buffer-2) to be reduced. The inclusion of a denaturant in the solution deforms the protein structure so that inner disulfides are available to the immobilized reductant. Without the presence of guanidine or another deforming agent (i.e., urea, SDS, etc.), only partial reduction of the protein is possible. 

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