(d) Sample/spiked sample/spike preparation.
l
Sample.
This solution must undergo deoxycholate precipitation described in the following sections.
– Label a 2 mL Eppendorf tube as “Sample.”
– Transfer 1440 μL of “Sample solution at 100 μg/
mL in NaCl 0.1 M” in the tube.
– Add to the tube 60 μL of “NaCl 0.1 M solution”
and homogenize its content by vortexing.
l
Spiked sample.
This solution must undergo to deoxycholate precipitation described in the following sections.
– Label a 2 mL Eppendorf tube as “spiked sample.”
– Transfer 1440 μL of “Sample at 100 μg/mL in
NaCl 0.1 M” in the tube.
– Add to the tube 60 μL of “OAg solution 700 μg/
mL” and homogenize its content by vortexing.
l
Spike.
This solution does not have to undergo the deoxycholate precipitation but will be assayed in HPAECPAD to quantify the OAg spiked quantity.
– Label a 2 mL Eppendorf tube as “Spike.”
– Transfer 1380 μL of NaCl 0.1 M solution in
the tube.
– Add to the tube 60 μL of “OAg solution 700 μg/
mL” and homogenize its content by vortexing.
Summary of volumes in Table 9.
(e) Deoxycholate precipitation.
l
Cool the two Eppendorf tubes “sample” and “spiked
sample” in ice for 30 min.
l
Add to each Eppendorf tube 0.15 mL of deoxycholate solution, vortex them for some seconds and put
in ice for 30 min again.
Remark: cooling in ice is extremely important.
l
Transfer 1 mL of hydrochloric acid (HCl) 1 M solution in a 2 mL Eppendorf tube and cool it in ice for at
least 10 min.
Remark: cooling in ice is extremely important.
l
Add to each Eppendorf tube 75 μL of cooled HCl
1 M solution, vortex them for some seconds and
centrifuge at 12,000 RCF for 30 min at 4
C.
O-Antigen Purification and Conjugation
299
l
Sample.
This solution must undergo deoxycholate precipitation described in the following sections.
– Label a 2 mL Eppendorf tube as “Sample.”
– Transfer 1440 μL of “Sample solution at 100 μg/
mL in NaCl 0.1 M” in the tube.
– Add to the tube 60 μL of “NaCl 0.1 M solution”
and homogenize its content by vortexing.
l
Spiked sample.
This solution must undergo to deoxycholate precipitation described in the following sections.
– Label a 2 mL Eppendorf tube as “spiked sample.”
– Transfer 1440 μL of “Sample at 100 μg/mL in
NaCl 0.1 M” in the tube.
– Add to the tube 60 μL of “OAg solution 700 μg/
mL” and homogenize its content by vortexing.
l
Spike.
This solution does not have to undergo the deoxycholate precipitation but will be assayed in HPAECPAD to quantify the OAg spiked quantity.
– Label a 2 mL Eppendorf tube as “Spike.”
– Transfer 1380 μL of NaCl 0.1 M solution in
the tube.
– Add to the tube 60 μL of “OAg solution 700 μg/
mL” and homogenize its content by vortexing.
Summary of volumes in Table 9.
(e) Deoxycholate precipitation.
l
Cool the two Eppendorf tubes “sample” and “spiked
sample” in ice for 30 min.
l
Add to each Eppendorf tube 0.15 mL of deoxycholate solution, vortex them for some seconds and put
in ice for 30 min again.
Remark: cooling in ice is extremely important.
l
Transfer 1 mL of hydrochloric acid (HCl) 1 M solution in a 2 mL Eppendorf tube and cool it in ice for at
least 10 min.
Remark: cooling in ice is extremely important.
l
Add to each Eppendorf tube 75 μL of cooled HCl
1 M solution, vortex them for some seconds and
centrifuge at 12,000 RCF for 30 min at 4
C.
O-Antigen Purification and Conjugation
299