Ziptip tips

Reactive Lys residues available for chemical coupling of antigens were determined by FITC-labeling free amino groups, FITC-modified coat protein was separated in SDS/PAGE gel, the fragment was excised from gel and digested with trypsin (Trypsin Profile IGD Kit, Sigma). VLPs (1.5 mg/ml in 50 mM Sodium borate, 2 mM EDTA, pH 9.0) were reacted with 20 mM FITC at +4 ^oC for 24 h. Samples after FITC treatment were separated in 12% SDS-PAGE, protein spots after Coomassie staining excized from gel and treated with Trypsin using Trypsin Profile IGD Kit and protocol (Sigma, St Louis, USA) ON +4 ^oC. The reaction mixture was purified using ZipTip tips (Merck Millipore, Cork, Ireland), diluted with a 3-hydroxypicolinic acid matrix solution and spotted onto an MTP AnchorChip 400/384TF. MALDI-TOF MS analysis was carried out on an Autoflex MS (Bruker Daltonik, Bremen, Germany). The protein molecular mass (MM) calibration standard I (3–20 kDa; Bruker Daltonik) and Peptide calibration standard II was used for mass determination.

Of the protein solutions, 27 µL were separated on precast 12% Criterion XT Bis−Tris gels (Biorad, Hercules, CA, USA). In-gel digestion was done according to Bonn et al. [48 (link)]. Sample gel lanes were cut into ten pieces, proteins digested in-gel with trypsin, and after elution from gel, desalting was performed with ZipTip-tips (Merck Chemicals GmbH, Darmstadt, Germany), according to manufacturer’s instructions. After drying samples at 30 °C in a vacuum centrifuge, peptides were resuspended in 10 µL 0.1% acetic acid in H₂O and transferred to glass vials.