Blue dextran

Pool of shrew serum from two animals (1 and 2) as: 3 × 150 μl animal 1 + 2 mixed, 3 × 150 μl animal 1 only, 3 × 150 μl animal 2 only were assayed as previously described (Gimsing and Nexø, 1989 (link)). Briefly, total B12 was measured using serum diluted 1:5 with 0.9% NaCl on the automatic platform Advia Centaur CP Immunoassay system. Unsaturated B12 binding capacity was measured on serum diluted 1:10 employing coated charcoal to separate free and bound ⁵⁷Co-B12 (KemEnTec) to separate TC and HC. A sample saturated with ⁵⁷Co-B12 was run on a Superdex® 200 HR 10/30 column (GE Healthcare Europe) using a Dionex® ICS-3000 chromatograph (Dionex Corporation), eluted at a flow rate of 400 μL/min for approx. 70 min employing a Tris buffer (0.1 mol/L) (Sigma Aldrich), 1 mol/L NaCl (Merck), 0.5 g/L bovine albumin (Sigma Aldrich), 0.2 g/L sodium-azide (Merck), pH 8.0. The eluted fractions were counted in a Wizard Automatic Gamma Counter to monitor the elution of ⁵⁷Co-B12. Molecular mass markers Blue Dextran (Sigma Aldrich) and ²²Na (GE Healthcare Europe) were used for determination of void volume (V_o), and total volume (V_t).

100 µM ΔlgtE LOS vesicles were incubated in 150 µL reaction buffer (20 mM HEPES pH 7.5, 1 mM MnCl2, 1 µM LgtE, 1 mM UDP-galactose), at 30 °C for a minimum of 2 h. The vesicle containing solution was applied to a column (length 5.5 cm, diameter 1.5 cm) containing Sepharose 2B (GE Health Sciences) for size exclusion chromatography. The samples were run with Blue Dextran (Sigma) as a void volume marker. Fractions (1 mL) were collected. Intact vesicles eluted primarily is fractions 2 and 3. To confirm the presence of the vesicles, 15 µ of each fraction was mixed with 5 µl 3x Laemmli buffer and then subjected to SDS-PAGE analysis. The fractions were run on a 16% Criterion™ Tris-Tricine Precast gel (Bio-Rad Laboratories Inc.). The gel was run at a constant current of 3 mAmp, on ice, for approximately 8 h. The gel was then fixed and silver-stained as described above.