S9640

Throughout this work, all immunoprecipitations were preceded by an in vitro antibody agarose bead crosslinking step, to prevent excessively abundant peptides from the antibodies which can generate unwanted interferences. Antibodies were incubated with Pierce Protein G Agarose beads (20398; Thermo Fisher Scientific) at a 1:20 ratio (5 μl of antibody for 100 μl of beads) for 1 h rocking. Antibody-bound beads (henceforth referred to simply as beads) were then washed with sodium tetraborate decahydrate (S9640; Sigma-Aldrich), 0.1 M, pH 9, and resuspended again in sodium tetraborate decahydrate with dimethyl pimelimidate (DMP) (21667; Thermo Fisher Scientific) at the final concentration of 20 mM. The DMP and beads were mixed for 30 min at RT, after which the reaction was terminated by washing the beads once with ethanolamine (E0135; Sigma-Aldrich) 0.2 M pH 8 and three times with PBS. Finally, beads were resuspended in PBS with 0.02% (wt/vol) sodium azide (S2002; Sigma-Aldrich) and stored at 4°C until use (no longer than 48 h).

HS amount in purified EDs or isolated HS chains was quantified as previously described in detail^{71 (link)}. Briefly, 150 µl/well of a 25 mM sodium tetraborate (Sigma-Aldrich #S9640) solution in 98% sulfuric acid were added to a 96-microplate kept on an ice-bed. To this, 40 µl/well of 1:10 H₂O-diluted samples of isolated syndecan EDs were added. The plate was transferred to a 100 °C oven for 15 min. After incubation, the plate was returned to the ice-bed, allowed to cool down and then 4 µl/well of a 0.125% w/v carbazole (Sigma Aldrich # 442506) solution in ethanol were added. The plate was then incubated at 100 °C for 10 additional minutes followed by absorbance measurement at 525 nm. Heparin (Sigma Aldrich # H3393) was used to build a standard curve for quantification.

Methylene blue (≥82%, #M9140), sodium borate (ACS reagent, ≥99.5%, #S9640) and chloroform (≥99.5%, #C2432) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rhamnolipid was quantified as described by Pinzon et al. [58 (link)]. The supernatant of the anti-biofilm test was collected, and bacterial cells were removed by centrifugation (KUBOTA, Osaka, Japan). The pH of the cell-free supernatant was first adjusted to 2.3 ± 0.2 using 1 N HCl. Two hundred microliters of acidified sample was then extracted with the same volume of chloroform five times. One milliliter of the chloroform extract was carefully removed and mixed with 40 μL of 0.1% Methylene blue (freshly prepared and pH adjusted to 8.6 ± 0.2 with 50 mM borate buffer) and 960 μL of distilled water in a 2 mL tube. After being vigorously shaken for 4 min, the samples were left to stand for 15 min. The chloroform phase was transferred into a quartz cuvette and value of A_{638 nm} was measured by spectrophotometer (U3310, HITACHI, Tokyo, Japan). The values were converted to rhamnolipid concentrations using a calibration curve established by applying the same procedure to standard rhamnolipid solutions of different concentrations.