Lane 1 – MW markers; Lane 2 – ab83547; Lane 3 – ab83547 treated with PNGase F to remove potential N-linked glycans; Lane 4 – ab83547 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 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 ab83547 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 isoforms of ab83547. Spots within the spot train were cut from the gel and identified as ab83547 by protein mass fingerprinting.
Post-translational modifications result in protein heterogeneity. The densitometry scan demonstrates that ab83547 exists in multiple isoforms, which differ according to their level of post-translational modification. Expression of these isoforms is highly significant for cell biology, as they more closely resemble the native human proteins. The triangle indicates theoretical pI and MW of the protein.