Bodipy fl hydrazide

A conjugate of oxidized S-LPS with the Alexa Fluor 647 fluorescent dye (AF-LPS) was prepared as follows. S-LPS was suspended at 1 mg/ml in 0.5 ml of 0.1 M sodium acetate (pH 5.0) and incubated with 1 mM sodium metaperiodate (Sigma-Aldrich) for 30 min on ice [33 (link)]. The reaction was stopped by adding 4 μl of glycerol and oxidized LPS was separated from other reagents by extensive, 24-h dialysis against PBS at 4°C (6–8 kDa cutoff). Fifty μl of Alexa Fluor 647 hydrazide (Invitrogen), dissolved at 50 mM in dimethyl sulfoxide, was added to 0.5 ml of LPS for 2-h incubation at room temperature. Unreacted dye was separated by dialysis. The estimated degree of labeling in this AF-LPS preparation was ~0.47:1 (AF:S-LPS).
To prepare a conjugate of native S-LPS with BODIPY FL (BO-LPS), S-LPS was suspended at 2 mg/ml in 0.25 ml of PBS, sonicated and mixed with 25 μl of 50 mM BODIPY FL hydrazide (Invitrogen) in dimethyl sulfoxide. After 40-min incubation at 37°C, the mixture was sonicated again, 0.2 ml of 0.2 M NaHCO_3, (pH 8.7) and another 25 μl of BODIPY FL hydrazide were added and the incubation was continued for further 1 h. The sample was then microfuged and the supernatant dialyzed. The estimated degree of labeling in BO-LPS was ~0.13:1.

HS was extracted and purified from individual cerebella in Trizol reagent (Invitrogen and [37 (link)]). The aqueous phase was applied to a DEAE column; HS was eluted with 2 M NaCl, and desalted on a PD10 column (GE Healthcare). Digestion reactions with recombinant heparinase enzymes (Ibex, Canada) from Flavobacterium heparinum were carried out at 37°C using initial digestion with heparinase I for 2 h, followed by heparinase III for 2 h and heparinase II overnight (16 hrs). Heparinase enzymes were used at 2.5mU in 10 μl heparinase buffer (100 mM sodium acetate, 0.1 mM calcium acetate, pH 7.0). HS disaccharides were labelled with Bodipy™ FL hydrazide (Invitrogen), separated by SAX-HPLC on a Propac PA-1 column (Dionex, UK), and detected in-line using a Shimadzu RF-551 fluorescence detector, as previously described [35 (link),36 (link),37 (link)]. Disaccharide peaks were identified by reference to the consistent elution positions of authentic disaccharide standards, and peak quantification performed using Shimadzu software with baseline subtraction, using previously calculated correction factors to account for differential labeling [52 (link)].