l
To each tube containing standards or samples add
200 μL of 5% phenol solution using the automatic
pipette equipped with a 10 mL disposable Combitips
syringe.
l
Vortex each tube for few seconds.
l
To each tube containing standards and samples, add
1 mL of sulfuric acid using the automatic pipette
equipped with a disposable 25 mL Combitips syringe
(avoid using normal pipettes because the sulfuric acid
vapor will damage their pistons) (see Note 21).
Fig. 3 (a) SDS-PAGE analysis of conjugation mixture in comparison to unconjugated carrier protein: lane 1:
marker, lane 2: CRM 197 (2 μg loaded), lane 3: Shigella flexneri 6 OAg-ADH-SIDEA-CRM 197 (10 μg loaded). (b)
HPLC-SEC profiles (fluorescence emission detection) of Shigella flexneri 6 OAg-ADH-SIDEA-CRM 197 (100 μg/
mL in protein) in comparison to free CRM 197 (100 μg/mL) and free OAg. 80 μL of each sample was run on
Tosoh TSK gel 6000 + 5000 PW columns; eluent 0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN, pH 7.2; flow rate
0.5 mL/min. Free OAg is not detected by fluorescence emission
Table 1
Dilutions for preparing the Glc calibration curve for phenol sulfuric assay
Std conc. (μg/mL)
Glc 0.5 mg/mL (μL)
Water (μL)
0
0
200
62.5
25
175
125
50
150
187.5
75
125
250
100
100
282
Francesca Micoli et al.
To each tube containing standards or samples add
200 μL of 5% phenol solution using the automatic
pipette equipped with a 10 mL disposable Combitips
syringe.
l
Vortex each tube for few seconds.
l
To each tube containing standards and samples, add
1 mL of sulfuric acid using the automatic pipette
equipped with a disposable 25 mL Combitips syringe
(avoid using normal pipettes because the sulfuric acid
vapor will damage their pistons) (see Note 21).
Fig. 3 (a) SDS-PAGE analysis of conjugation mixture in comparison to unconjugated carrier protein: lane 1:
marker, lane 2: CRM 197 (2 μg loaded), lane 3: Shigella flexneri 6 OAg-ADH-SIDEA-CRM 197 (10 μg loaded). (b)
HPLC-SEC profiles (fluorescence emission detection) of Shigella flexneri 6 OAg-ADH-SIDEA-CRM 197 (100 μg/
mL in protein) in comparison to free CRM 197 (100 μg/mL) and free OAg. 80 μL of each sample was run on
Tosoh TSK gel 6000 + 5000 PW columns; eluent 0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN, pH 7.2; flow rate
0.5 mL/min. Free OAg is not detected by fluorescence emission
Table 1
Dilutions for preparing the Glc calibration curve for phenol sulfuric assay
Std conc. (μg/mL)
Glc 0.5 mg/mL (μL)
Water (μL)
0
0
200
62.5
25
175
125
50
150
187.5
75
125
250
100
100
282
Francesca Micoli et al.