2 Materials
Prepare all solutions using ultrapure water (grade 1, >18 MΩ-cm at
25
C; prepared by purifying deionized water) and analytical grade
reagents. Prepare and store all reagents at room temperature (RT),
filter all the solutions using a 0.22 μm filtration system (e.g., Merck
Stericup), unless otherwise indicated.
2.1 OAg purification
1. NaCl 1 M: Add about 900 mL water in a 1 L graduated
cylinder equipped with a magnetic stir bar. Weigh 58.44 g
NaCl and transfer into the cylinder. Mix on a magnetic stirrer
(see Note 1) until all the powder is dissolved. Add water up to
1 L total volume and mix again.
2. Citrate buffer 200 mM pH 2.7 (see Note 2): Weigh 28.9 g
citric acid monohydrate and 13.3 g sodium citrate monobasic
and transfer into a cylinder filled with about 900 mL water. Mix
until the powder is completely dissolved; adjust the pH if
needed (see Note 3) and add water up to 1 L total volume.
3. Citrate buffer 20 mM pH 3: Weigh 2.3 g citric acid monohydrate and 1.9 g sodium citrate monobasic and prepare a 1 L
buffer solution, as described in item 2.
Fig. 1 O-antigen isolation through mild acetic acid hydrolysis: the labile linkage
between KDO, at the proximal end of the core, and lipid A is cleaved releasing
the O-antigen chain attached to the core sugars
O-Antigen Purification and Conjugation
269
Prepare all solutions using ultrapure water (grade 1, >18 MΩ-cm at
25
C; prepared by purifying deionized water) and analytical grade
reagents. Prepare and store all reagents at room temperature (RT),
filter all the solutions using a 0.22 μm filtration system (e.g., Merck
Stericup), unless otherwise indicated.
2.1 OAg purification
1. NaCl 1 M: Add about 900 mL water in a 1 L graduated
cylinder equipped with a magnetic stir bar. Weigh 58.44 g
NaCl and transfer into the cylinder. Mix on a magnetic stirrer
(see Note 1) until all the powder is dissolved. Add water up to
1 L total volume and mix again.
2. Citrate buffer 200 mM pH 2.7 (see Note 2): Weigh 28.9 g
citric acid monohydrate and 13.3 g sodium citrate monobasic
and transfer into a cylinder filled with about 900 mL water. Mix
until the powder is completely dissolved; adjust the pH if
needed (see Note 3) and add water up to 1 L total volume.
3. Citrate buffer 20 mM pH 3: Weigh 2.3 g citric acid monohydrate and 1.9 g sodium citrate monobasic and prepare a 1 L
buffer solution, as described in item 2.
Fig. 1 O-antigen isolation through mild acetic acid hydrolysis: the labile linkage
between KDO, at the proximal end of the core, and lipid A is cleaved releasing
the O-antigen chain attached to the core sugars
O-Antigen Purification and Conjugation
269