Zebra desalting columns

To evaluate the effect of R17 and its variants on the ability of CCL2 to interact with cell surfaces, chemokines and a negative control protein (MR1) were non-specifically biotinylated using an EZ-biotin kit (Pierce) with a 2:1 biotin to protein molar ratio, followed by removal of unbound biotin (Thermo Scientific Zebra Desalting Columns). CHOK1 and CHO745 cells were maintained in F-12 media supplemented with 10% FCS and 100X PennStrep. On the day of the experiment, cells were washed once with PBS, detached using 0.2% EDTA and re-suspended in staining buffer containing PBS, 1% BSA and 2mM EDTA. Biotinylated CCL2 was added to cells at a final concentration of 50nM in the presence of absence of R17^GAG1 or R17^GAG2, incubated for an hour on ice, washed three times and detected with Streptavidin PE (Life Technologies) using a FACSCalibur (BD biosciences).

Single cysteine mutants of LPL were purified (final buffer composition was 1.5 M NaCl, 10% glycerol, 20 mM Bis-Tris pH 6.5) and incubated with an approximately 10-fold excess of Alexa Fluor® Maleimide Dyes (Invitrogen). Label was added dropwise to the protein solution followed by gentle mixing. The reaction was allowed to continue, in light protected tubes, for 45 minutes at 4 °C or on ice. For samples in which biotinylated LPL was used, a 1–2 molar excess of Biotin-N-Hydroxysuccinimide ester (Sigma) was next added and the reaction was allowed to proceed on ice for another 10 minutes. For both biotinylated and non-biotinylated samples, LPL was then diluted to less than 500 mM NaCl with 20 mM Bis-Tris pH 6.5 and re-purified over a HiTrap Heparin Sepharose High Performance Column (GE Healthcare Life Sciences) to remove excess biotin and/or label. Excess label flowed through the heparin column whereas purified, labeled LPL bound and eluted normally. Alternatively, labeling could be followed by removal of excess dye by use of two tandem 7 kDa Zebra desalting columns (ThermoFisher Scientific). LPL samples were aliquoted and stored at −80 °C until use. If samples were concentrated, the samples were syringe filtered through a 0.2 μm PDVF filter to remove aggregates.