4. Citrate buffer 20 mM pH 3, NaCl 1 M: Weigh 2.3 g citric acid
monohydrate, 1.9 g sodium citrate monobasic and 58.44 g
NaCl and prepare a 1 L buffer solution, as described in item 2.
5. Sodium phosphate monobasic (NaH 2 PO 4 ) 500 mM: Weigh
60 g NaH 2 PO 4 and prepare a 1 L solution.
6. Calcium chloride (CaCl 2 ) 5 M: Weigh 554.9 g CaCl 2 and
prepare a 1 L solution.
7. Tangential flow filtration (TFF) 30 KDa Sartorius Hydrosart
200 cm
2 membrane (stabilized cellulose-based membrane).
8. Sartobind S MA75 filter (housing polysulfone membrane
matrix stabilized cellulose, nominal pore size >3 μm strong
cation exchanger, membrane area 75 cm
2 ).
2.2 OAg Conjugation
to Carrier Protein
and Conjugate
Purification
1. Acetate buffer 100 mM pH 4.5: Withdraw 3.4 mL acetic acid
and 3.3 g sodium acetate (AcONa) and prepare a 1 L buffer
solution, as described in Subheading 2.1, item 2.
2. Citrate buffer 100 mM pH 3: Weigh 11.1 g citric acid monohydrate and 10 g sodium citrate monobasic and prepare a 1 L
buffer solution, as described in Subheading 2.1, item 2.
3. Phosphate buffer 100 mM pH 7.2: Weigh 3.2 g NaH 2 PO 4 and
10.4 g sodium phosphate dibasic (Na 2 HPO 4 ) and prepare a
1 L buffer solution, as described in Subheading 2.1, item 2.
4. Phosphate buffer 50 mM pH 7.2, NaCl 150 mM: Weigh 1.8 g
NaH 2 PO 4 , 4.9 g Na 2 HPO 4 , and 8.8 g NaCl and prepare a 1 L
buffer solution, as described in Subheading 2.1, item 2.
5. 16/90 cm Sephacryl S-300 HR column.
2.3 Analytical
Methods
1. HiTrap desalting column, 5 mL, prepacked with Sephadex
G-25 Superfine (GE Healthcare).
2. Phenol sulfuric colorimetric method.
(a) 5% Phenol solution.
l
Weigh on technical balance a 100 mL glass bottle with
its screw cap and set it as tare.
l
Under chemical hood transfer some phenol in the
bottle and close it.
l
Weigh on the technical balance (tared with the empty
bottle and cap) the amount of phenol transferred in the
bottle.
l
Repeat operations described in the previous sections in
order to reach a quantity close to 5 g.
l
Calculate the quantity of water needed to obtain a 5%
(w/v) phenol solution using the following formula:
Water to add (mL) ¼ phenol weight (g) Â 20.
270
Francesca Micoli et al.
monohydrate, 1.9 g sodium citrate monobasic and 58.44 g
NaCl and prepare a 1 L buffer solution, as described in item 2.
5. Sodium phosphate monobasic (NaH 2 PO 4 ) 500 mM: Weigh
60 g NaH 2 PO 4 and prepare a 1 L solution.
6. Calcium chloride (CaCl 2 ) 5 M: Weigh 554.9 g CaCl 2 and
prepare a 1 L solution.
7. Tangential flow filtration (TFF) 30 KDa Sartorius Hydrosart
200 cm
2 membrane (stabilized cellulose-based membrane).
8. Sartobind S MA75 filter (housing polysulfone membrane
matrix stabilized cellulose, nominal pore size >3 μm strong
cation exchanger, membrane area 75 cm
2 ).
2.2 OAg Conjugation
to Carrier Protein
and Conjugate
Purification
1. Acetate buffer 100 mM pH 4.5: Withdraw 3.4 mL acetic acid
and 3.3 g sodium acetate (AcONa) and prepare a 1 L buffer
solution, as described in Subheading 2.1, item 2.
2. Citrate buffer 100 mM pH 3: Weigh 11.1 g citric acid monohydrate and 10 g sodium citrate monobasic and prepare a 1 L
buffer solution, as described in Subheading 2.1, item 2.
3. Phosphate buffer 100 mM pH 7.2: Weigh 3.2 g NaH 2 PO 4 and
10.4 g sodium phosphate dibasic (Na 2 HPO 4 ) and prepare a
1 L buffer solution, as described in Subheading 2.1, item 2.
4. Phosphate buffer 50 mM pH 7.2, NaCl 150 mM: Weigh 1.8 g
NaH 2 PO 4 , 4.9 g Na 2 HPO 4 , and 8.8 g NaCl and prepare a 1 L
buffer solution, as described in Subheading 2.1, item 2.
5. 16/90 cm Sephacryl S-300 HR column.
2.3 Analytical
Methods
1. HiTrap desalting column, 5 mL, prepacked with Sephadex
G-25 Superfine (GE Healthcare).
2. Phenol sulfuric colorimetric method.
(a) 5% Phenol solution.
l
Weigh on technical balance a 100 mL glass bottle with
its screw cap and set it as tare.
l
Under chemical hood transfer some phenol in the
bottle and close it.
l
Weigh on the technical balance (tared with the empty
bottle and cap) the amount of phenol transferred in the
bottle.
l
Repeat operations described in the previous sections in
order to reach a quantity close to 5 g.
l
Calculate the quantity of water needed to obtain a 5%
(w/v) phenol solution using the following formula:
Water to add (mL) ¼ phenol weight (g) Â 20.
270
Francesca Micoli et al.