Journal: bioRxiv

Article Title: Topological reprogramming transforms an integral membrane oligosaccharyltransferase into a water-soluble glycosylation catalyst

doi: 10.64898/2026.01.30.702934

Figure Lengend Snippet: (a) Schematic of TAMRA peptide IVG assay. Incubation of TAMRA-labeled peptides with OSTs, Cj LLOs, and divalent metal ions (e.g., Mn 2+ ) results in transfer of heptasaccharide glycans to form glycopeptide products. (b) Tricine SDS-PAGE analysis of IVG reactions with different amounts of TAMRA-labeled peptide and 0.5 μM of each OST. Graph at right depicts determination of Michaelis–Menten kinetics using IVG reaction data. Data fitting was by nonlinear regression analysis according to Michaelis–Menten model using Prism 10 for MacOS (version 10.3.0). Black arrows denote aglycosylated (g0) and singly glycosylated (g1) forms of TAMRA-labeled peptides. Tricine SDS-PAGE gels in each panel are representative of three biological replicates. Data in corresponding graphs are average of biological replicates ( n = 3) ± SD.

Article Snippet: Protein samples were denatured by addition of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer containing 10% β-mercaptoethanol, followed by heating at 90 °C for 10 min. Electrophoretic separation was performed using either precast 4–20% Tris-Glycine gels (Invitrogen) or AnyKD Mini-PROTEAN TGX gels (Bio-Rad).

Techniques: Incubation, Labeling, Glycoproteomics, SDS Page