Since SDS displayed several additional, radiolabeled protein bands, immunoprecipitated VE-cadherin samples were preparatively separated by SDS-PAGE, and bands containing VE-cadherin were excised (I in Figure 1)
In order to avoid loss of material when eluting the whole glycoprotein , an in-gel proteolytic digestion was performed, after which radioactive glycopeptides could be easily eluted from the gel pieces
From the known amino acid sequence of the glycoprotein (Suzuki ; Breviario et al., 1995), it could be deduced that treatment with trypsin would generate a glycopeptide resistant to peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F ( PNGase F) (Tarentino et al., 1985)
Therefore, Asp-N endoproteinase from Pseudomonas fragi mutant was used instead
In total, a glycopeptide preparation was obtained comprising about 105 cpm of incorporated 3H radioactivity
As a control, unstained gel segments (II in Figure 1) were equally treated and eluted
However, no radioactivity could be detected in the respective supernatants