Mercaptoethanol disulfide preparation

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Sodium dodecyl sulfate (SDS) gel electrophoresis systems are used to determine the number and size of protein chains or protein subunit chains in a protein preparation. Initially, the protein preparation is treated with an excess of soluble thiol (usually 2-mercaptoethanol) and SDS. Under these conditions, the thiol reduces all disulfide bonds (-S-S-) present within and/or between peptide units, while the SDS (an ionic or... 

Preparation of sample and buffers Protein is prepared from the acquired sample using conventional protein extraction techniques, and treated with SDS to denature the protein, and commonly the protein is also exposed to a reducing agent. Reducing agents, such as dithiothreitol or 2-mercaptoethanol, break (or reduce) the disulfide bonds in the protein and, if used, the method is called... 

In the O Farrell method, SDS is used in the second dimension, -mercaptoethanol in the first. Others have used SDS in both dimensions and in sample preparation. The use of p-mercaptoethanol and SDS denatures proteins to polypeptides by reducing disulfide bonds and depolymeriz-ing proteins. When native proteins, such as enzymes, are desired for further study, nondenaturing sample preparation and electrophoresis conditions must be used. 

PROTECTING GROUPS - Cyclic imide disulfides from primary amines can be prepared by the scheme below. Treatment with mercaptoethanol under very mild conditions gives back the starting amine. 

The thiols, mercaptoethanol and diethiothreitol, have been used extensively for the reductive cleavage of disulfide bonds and for the maintenance of a reductive environment in the isolation and characterization of specific soybean proteins (2). However, these reagents have not been used for the preparation of protein products for food. The odor and flavor of the low-molecular-weight thiol compounds are not compatible with usage in a broad range of food items, yet these substances contribute to the desirable odor and flavor of selected foods. 

The next step in sample preparation for MS analysis is reduction of intramolecular disulfide bonds by using reducing agents, such as dithiothreitol (DTT), dithioerythritol (DTE), tributylphosphine (TBP), tris(2-carboxyethyl)phosphine (TCEP), or P-mercaptoethanol (BME). The reduced disulfide bonds should be blocked by alkylatiou to prevent refolding. For this type of reaction, the typical procedures iuvolve carbamidomethylation of cysteines with iodoacetamide or pyridylethylation. 

Half-molecules are prepared from rabbit IgG by taking advantage of two properties of the molecule. First, the disulfide bond joining the two H chains (H-H bond) is more labile than the H-L bonds and, in fact, is the most labile disulfide bond in the molecule in the presence of 0.01 M 2-mercaptoethanol nearly aU H-H disulfide bonds, but very few H-L bonds, are reduced (56). Second, the noncovalent forces joining the two H chains are weaker than those linking H and L chains. Thus, even if all interchain disulfide bonds are reduced and alkylated, and the molecule is then exposed to HCl solution at pH 2.5, half-molecules, rather than free H and L chains, are liberated because the noncovalent interactions between H and L chains are not disrupted (56). (Separation of H from L chains requires more drastic dissociating conditions.) In practice, then, half-molecules are produced by reduction with 0.01 M to 0.02 M 2-mercaptoethanol, followed by alkylation of sulfhydryl groups and exposure to HCl-NaCl, pH 2.5 (54,55). The separation can be monitored in the... 

---