Lane 1 – MW markers; Lane 2 – ab84062; Lane 3 – ab84062 treated with PNGase F to remove potential N linked glycans; Lane 4 – ab84062 treated with a glycosidase cocktail to remove potential N- and O-linked glycans. Approximately 5 μg of protein was loaded per lane; Gel was stained using Coomassie. Drop in MW after treatment with PNGase F indicates the presence of N-linked glycans. A further drop in MW after treatment with the glycosidase cocktail indicates the presence of O-linked glycans. Additional bands in lane 3 and lane 4 are glycosidase enzymes.
A sample of ab84062 without carrier protein was reduced and alkylated and focused on a 3-10 IPG strip then run on a 4 20% Tris-HCl 2D gel. Approximately 40 μg of protein was loaded; Gel was stained using Deep Purple™. The spot train indicates the presence of multiple glycoforms. Spots within the spot train were cut from the gel and identified as Cripto1 (Fc Chimera) by protein mass fingerprinting.
Post-translational modifications result in protein heterogeneity. The densitometry scan demonstrates the purified human cell expressed protein exists in multiple glycoforms, which differ according to their level of post-translational modification. The triangle indicates theoretical pI and MW of the protein.