l
Sodium acetate (AcONa) 1 M NaOH 100 mM.
– Degas 1.5 L of water for 15 min by bubbling Helium.
– Add, directly in the AcONa container, about 500 mL of
degassed water and dissolve the whole content of
AcONa (Thermo Dionex, cod. 059326). Transfer the
solution in the 1 L volumetric flask.
– Wash twice the AcONa bottle with about 100 mL more
of degassed water and add the solution to the 1 L
volumetric flask.
– Fill the 1 L volumetric flask with degassed water, up to
1 L of volume for the AcONa solution.
– Place a 0.22 μm vacuum-cup filter on an empty eluent
2 L bottle, connect it to the vacuum system and filter
the prepared 1 L solution.
– Degas the solution for 10 min by bubbling Helium.
– With a pipette, add 5.2 mL of NaOH 50% to the bottle.
– Degas with Helium for 10 min more.
4. Micro BCA.
(a) Thermo micro BCA Protein Assay kit (BSA calibration
curve range: 5–20 μg/mL).
5. Charles River Endosafe-PTS instrument cartridges for chromogenic kinetic LAL (Limulus amoebocyte lysate).
6. Size-exclusion high-performance liquid chromatography
(HPLC-SEC).
(a) Tosoh TSK gel G3000 PWXL column (30 cm  7.8 mm;
cod. 808021) with a Tosoh TSK gel PWXL guard column
(4.0 cm  6.0 mm; cod. 808033).
(b) Tosoh TSK gel 6000 PW column (30 cm  7.5 mm; cod.
805765) connected in series with a Tosoh TSK gel 5000
PW column (30 cm  7.5 mm; cod. 805764) with Tosoh
TSK gel PWH guard column (7.5 cm x 7.5 mm; cod.
806732).
(c) 0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN, pH 7.2.
l
Weigh 5.85 g of NaCl and put the powder inside the
1 L glass graduated cylinder.
l
Weigh 3.22 g of NaH 2 PO 4 and put the powder inside
the 1 L glass graduated cylinder.
l
Weigh 10.37 g of Na 2 HPO 4 and put the powder inside
the 1 L glass graduated cylinder.
l
Add water up to 850 mL in the 1 L graduated cylinder.
Put a magnetic stir bar inside the cylinder and solubilize the powder placing the cylinder on a magnetic
stirrer.
O-Antigen Purification and Conjugation
273
Sodium acetate (AcONa) 1 M NaOH 100 mM.
– Degas 1.5 L of water for 15 min by bubbling Helium.
– Add, directly in the AcONa container, about 500 mL of
degassed water and dissolve the whole content of
AcONa (Thermo Dionex, cod. 059326). Transfer the
solution in the 1 L volumetric flask.
– Wash twice the AcONa bottle with about 100 mL more
of degassed water and add the solution to the 1 L
volumetric flask.
– Fill the 1 L volumetric flask with degassed water, up to
1 L of volume for the AcONa solution.
– Place a 0.22 μm vacuum-cup filter on an empty eluent
2 L bottle, connect it to the vacuum system and filter
the prepared 1 L solution.
– Degas the solution for 10 min by bubbling Helium.
– With a pipette, add 5.2 mL of NaOH 50% to the bottle.
– Degas with Helium for 10 min more.
4. Micro BCA.
(a) Thermo micro BCA Protein Assay kit (BSA calibration
curve range: 5–20 μg/mL).
5. Charles River Endosafe-PTS instrument cartridges for chromogenic kinetic LAL (Limulus amoebocyte lysate).
6. Size-exclusion high-performance liquid chromatography
(HPLC-SEC).
(a) Tosoh TSK gel G3000 PWXL column (30 cm  7.8 mm;
cod. 808021) with a Tosoh TSK gel PWXL guard column
(4.0 cm  6.0 mm; cod. 808033).
(b) Tosoh TSK gel 6000 PW column (30 cm  7.5 mm; cod.
805765) connected in series with a Tosoh TSK gel 5000
PW column (30 cm  7.5 mm; cod. 805764) with Tosoh
TSK gel PWH guard column (7.5 cm x 7.5 mm; cod.
806732).
(c) 0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN, pH 7.2.
l
Weigh 5.85 g of NaCl and put the powder inside the
1 L glass graduated cylinder.
l
Weigh 3.22 g of NaH 2 PO 4 and put the powder inside
the 1 L glass graduated cylinder.
l
Weigh 10.37 g of Na 2 HPO 4 and put the powder inside
the 1 L glass graduated cylinder.
l
Add water up to 850 mL in the 1 L graduated cylinder.
Put a magnetic stir bar inside the cylinder and solubilize the powder placing the cylinder on a magnetic
stirrer.
O-Antigen Purification and Conjugation
273