Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(9)
Analysis of the Epitope Recognized by 2D6 McAb (Protein versus Carbohydrate)
The cauda form of 2D6 antigen is highly glycosylated, and since many monoclonal antibodies recognize specific sugar epitopes, a preliminary analysis was carried out to determine whether 2D6 monoclonal antibody was binding to protein or carbohydrate moieties. For this purpose a Western blot containing reduced proteins extracted from washed cauda sperm was incubated at 37°C for 16 h in 5 mM sodium phosphate, pH 7.6, containing 0.1% (v/v) Triton X-100, 1 mM Pefabloc, and 10 U/ml of Л-glycanase to cleave Л-linked carbohydrate chains. more
A parallel control was incubated under the same conditions but without the enzyme. Blots were washed in several changes of PBS followed by probing with either 2D6 McAb or 2D6 polyclonal antibody as described previously.
O-linked carbohydrates were released by alkaline hydrolysis as described by Duk et al. but with the omission of the preliminary acid step, as it appeared to cause degradation of the antigen. Western blots containing reduced cauda sperm proteins, previously separated by SDS-PAGE, were immersed in 55 mM NaOH, pH 12.0, at 4°C for various times up to 1 h, washed three times in PBS, and probed with 2D6 McAb and 2D6 polyclonal antibodies as described previously. As a positive control, parallel blots were probed with 2B1 McAb, which recognizes a peptide-based epitope.