sds page gel  (Bio-Rad)

Journal: bioRxiv

Article Title: An octadecameric O- glucosyltransferase generates diversity in antibody epitopes on variant surface antigens in African trypanosomes

doi: 10.64898/2026.01.27.701950

Figure Lengend Snippet: A. Purification. Coomassie-stained SDS-PAGE of affinity-purified recombinant ESAG3 (residues 23–368 with C-terminal His₁₀-EPEA tag) expressed in Expi293F cells. Arrow indicates ESAG3 monomer (∼50 kDa). M, molecular weight markers. B. UDP-Glo™ assay schematic. ESAG3-catalyzed glucosylation releases UDP from UDP-glucose, which is converted to ATP by UDP-Glo™ reagent, generating luminescence proportional to glycosyltransferase activity. Parallel mass spectrometry validates glucose addition. C. UDP-sugar donor specificity screen without acceptor. Purified ESAG3 was incubated with indicated UDP-sugar donors (1 hr, 30°C) and UDP release quantified by luminescence. ESAG3 hydrolyses UDP-glucose specifically (68-fold above background), while UDP-galactose (Gal), UDP-GlcNAc, UDP-GalNAc, and UDP-glucuronic acid (GA) show negligible activity. UDP-xylose shows modest activity (8-fold above background). Single experiment; RLU, relative light units. D. VSG peptide acceptor specificity. ESAG3 was incubated with indicated UDP-sugar donors and synthetic VSG-derived peptides (sequences shown; putative O-glucosylation sites S/T in blue). With UDP-glucose, all VSG peptides show 2.7–4-fold enhanced activity over no-acceptor baseline. UDP-galactose shows negligible activity. VSG3 (S317A,S319) peptide lacks S317 but retains S319 and T315 as potential modification sites. Y-axis: UDP-Glo luminescence (RLU × 10⁴). Bars show mean ± range (n=2 technical replicates). E. Metal cofactor requirement. ESAG3 activity with VSG3 (S317A,S319) peptide and UDP-glucose requires Mn²⁺. Mn²⁺ alone or combined with Mg²⁺ provides optimal activity; Mg²⁺ alone or omission of divalent cations shows only baseline activity. Bars show mean with individual replicates overlaid (n=3). F. MALDI-TOF MS confirms glucosylation. Analysis of VSG3 (S317A,S319) peptide DD-ATGCTGAASEGLCVEY-KK-NH₂ (expected mass 2014.9 m/z with disulfide bond). Left: Peptide-only control, no ESAG3. Middle: Reaction lacking UDP-glucose. Right: Complete reaction generates glucosylated product with +162.03 Da mass increase (blue annotation), consistent with hexose addition. Unmodified peptide indicated by red dotted line. Site-specific modification confirmed by LC-MS/MS ( and S3 ). G. LC-MS/MS confirms site-specific glucosylation at S319. Representative LC-MS/MS spectrum showing +162.05 Da modification on VSG3 (S317A,S319) peptide. Annotated fragment ions (b-ions in green, y-ions in orange) localise hexose to serine 11 of the peptide sequence (S11, corresponding to S319 in full-length VSG3). Site localisation is supported by the y₁₃ ion series (singly and doubly charged) that retains the +162 Da mass shift. This modification was detected in samples containing ESAG3 and UDP-glucose. Additional modification at threonine 7 (T7, corresponding to T315 in full-length VSG3) was also detected across replicates, with alternative site assignments detailed in Fig. S3 . Expected mass accounts for C-terminal amidation (–0.984 Da) from peptide synthesis.

Article Snippet: Samples were separated on 10% SDS-PAGE gels (Bio-Rad TGX FastCast Acrylamide Kit) or precast 4-12% NuPAGE Bis-Tris gels (ThermoFisher).

Techniques: Purification, Staining, SDS Page, Affinity Purification, Molecular Weight, Glo Assay, Activity Assay, Mass Spectrometry, Incubation, Derivative Assay, Modification, Control, Liquid Chromatography with Mass Spectroscopy, Sequencing