Urea can inhibit efficient reduction and alkylation of protein dimers in solution demonstrated by the beta subunit of alpha glucosidase II

Abstract

Protein reduction and alkylation is routinely used for analysis of protein dimers and protein complexes in cell fractions using two dimensional gel electrophoresis and mass spectrometry. To resolve the heterogeneity of a high molecular weight protein band that is highlighted by an antibody to the beta subunit of alpha glucosidase II (GIIβ), we performed reduction and alkylation of cytosolic proteins extracted from mouse brain. The presence of urea in the reduction/alkylation buffer inhibited the chemical processes. It is thus recommended that protein reduction/alkylation be performed both in the presence and absence of urea for the separation of mono-/hetero-mers.