Identification of mucins by using a method involving a combination of on-membrane chemical deglycosylation and immunostaining

Abstract

The characterization of mucins is critically important for gaining insights into the molecular pathology of diseases, including cancers, as well as for the discovery of biomarkers for disease diagnosis. However, no practical method has yet been reported for identifying mucin proteins. Here, we report a technique for immunological identification of mucins separated by supported molecular matrix electrophoresis (SMME), a recently developed membrane electrophoresis method. The technique involves on-membrane deglycosylation of mucins by using mild periodate oxidation/base-catalyzed elimination, followed by immunostaining with an antibody that specifically recognizes the mucin tandem repeat (TR) peptide. We demonstrated the method's feasibility by using MUC1 derived from 2 cancer cell lines, T47D and HPAF-II. The present method shows potential as an alternative approach for the identification of mucins separated by SMME or blotted from conventional gel electrophoresis, on a PVDF membrane.

Keywords: Antibody; Chemical deglycosylation; Identification; Mucins; On-membrane; SMME; Supported molecular matrix electrophoresis; TFMSA; TR; supported molecular matrix electrophoresis; tandem repeat; trifluoromethanesulfonic acid.