Lee, Der-YenDer-YenLeeChang, Geen-DongGeen-DongChang2010-10-072018-07-062010-10-072018-07-06200900032700http://ntur.lib.ntu.edu.tw//handle/246246/212603https://www.scopus.com/inward/record.uri?eid=2-s2.0-66149098167&doi=10.1021%2fac900281n&partnerID=40&md5=6ec4e0c0f7fc1d0a6936f06d1b2d8b6dArtifacts in two-dimensional electrophoresis (2-DE) caused by the presence of salts in isoelectric focusing (IEF) have been previously described as a result of increasing conductivity and inducing electroosmosis. However, electrolysis induced by the presence of salts should not be disregarded. In this study, electrolytic reduction-oxidation reaction (redox) was found to be enhanced in the presence of salts in IEF. The consequence of the electrolytic redox leads to acidification of the low-pH region and alkalization of the high-pH region within the immobilized pH gradient (IPG) strip. As a result, a breakdown of immobilized pH buffer near the high pH region of IPG strips along with reduction of basic proteins resulted in uncharacterized artifacts in 2-DE. Electrolytic reduction in the presence of alkali and alkaline metal ions was demonstrated to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein disulfide bonds, and protein carboxylic acids. Importantly, semipreparative electrolytic reduction of proteins can be carried out in the presence of sodium ions in a homemade electrolytic apparatus. These findings give additional explanations to the observed artifacts in 2-DE and reveal the unknown effects of salts in IEF. Moreover, we have provided a method with the potential to convert proteins or peptides to corresponding modified products containing aldehyde groups that can be used for conjugation with amine-containing compounds. © 2009 American Chemical Society.en-USAlkaline metal ions; Disulfide bonds; High pH; Isoelectric focusing; Modified products; pH buffers; pH gradients; Reduction-oxidation; Sodium ions; Two-dimensional electrophoresis (2-DE); Aldehydes; Alkalinity; Amines; Carboxylic acids; Electrolysis; Electrolytic reduction; Metal ions; Organic acids; pH effects; Proteins; Salts; Electrophoresis; amine; protein; sodium chloride; article; isoelectric focusing; oxidation reduction reaction; pH measurement; two dimensional electrophoresis; Amino Acid Sequence; Animals; Cattle; Electrophoresis, Gel, Two-Dimensional; Embryo, Nonmammalian; Isoelectric Focusing; Molecular Sequence Data; Oxidation-Reduction; Proteins; Proton-Motive Force; Ribonuclease, Pancreatic; Salts; Time Factors; Zebrafishjournal article194382642-s2.0-66149098167http://ntur.lib.ntu.edu.tw/bitstream/246246/212603/-1/03.pdf