l
Transfer the gel into the reservoir for staining containing 30 mL of water. Put on the gyro-rocker and
leave it rocking for about 2 min.
l
Remove water and add about 30 mL of Bio-Safe
Coomassie (mix the tank before use), being sure to
cover all the gel.
l
Leave the gel on the gyro-rocker for about 30 min.
l
Remove the stain solution and add 30 mL of water.
Rock the gel until bands are clear.
l
Collect a picture of the gel with a camera or scanner.
(f) Data evaluation.
l
Check conjugate formation by the presence of the
smear pattern typical of a glycoconjugate, in comparison to free protein band (Fig. 3a).
12. Free saccharide quantification.
The separation of free saccharide from conjugate is
achieved by conjugate coprecipitation with deoxycholate
[23]. Deoxycholate is added as sodium salt to the conjugate
solution containing sodium chloride 0.1 M; then lowering
the pH with HCl, the surfactant becomes insoluble and
coprecipitates with the conjugate. The free saccharide is
then quantified, for example, using HPAEC-PAD preparing
calibration curves in NaCl 0.1 M solution to reproduce sample matrix (as it affects the detector response).
In parallel to the sample above, a sample spiked with a
known amount of free OAg is assayed each time in order to
verify the procedure (spike recovery needs to be in the range
80–120%)
(a) OAg solution 700 μg/mL.
l
Dilute OAg to about 700 μg/mL in NaCl 0.1 M.
(b) Sample solution at 100 μg/mL in NaCl 0.1 M.
l
Prepare at least 4 mL of sample solution at about
100 μg/mL of saccharide (dilute it with water).
l
Label a 15 mL flacon tube with “Sample name
100 μg/mL NaCl 0.1 M.”
l
Weight inside the tube 24 Æ 1 mg of sodium chloride.
l
Transfer in the tube 4 mL of the sample solution
diluted to 100 μg/mL and mix on vortex to dissolve
all the salt.
(c) Free saccharide separation.
l
Initial Equipment preparation.
– Switch on the centrifuge and set the temperature
to 4
C to let it cool.
298
Francesca Micoli et al.
Transfer the gel into the reservoir for staining containing 30 mL of water. Put on the gyro-rocker and
leave it rocking for about 2 min.
l
Remove water and add about 30 mL of Bio-Safe
Coomassie (mix the tank before use), being sure to
cover all the gel.
l
Leave the gel on the gyro-rocker for about 30 min.
l
Remove the stain solution and add 30 mL of water.
Rock the gel until bands are clear.
l
Collect a picture of the gel with a camera or scanner.
(f) Data evaluation.
l
Check conjugate formation by the presence of the
smear pattern typical of a glycoconjugate, in comparison to free protein band (Fig. 3a).
12. Free saccharide quantification.
The separation of free saccharide from conjugate is
achieved by conjugate coprecipitation with deoxycholate
[23]. Deoxycholate is added as sodium salt to the conjugate
solution containing sodium chloride 0.1 M; then lowering
the pH with HCl, the surfactant becomes insoluble and
coprecipitates with the conjugate. The free saccharide is
then quantified, for example, using HPAEC-PAD preparing
calibration curves in NaCl 0.1 M solution to reproduce sample matrix (as it affects the detector response).
In parallel to the sample above, a sample spiked with a
known amount of free OAg is assayed each time in order to
verify the procedure (spike recovery needs to be in the range
80–120%)
(a) OAg solution 700 μg/mL.
l
Dilute OAg to about 700 μg/mL in NaCl 0.1 M.
(b) Sample solution at 100 μg/mL in NaCl 0.1 M.
l
Prepare at least 4 mL of sample solution at about
100 μg/mL of saccharide (dilute it with water).
l
Label a 15 mL flacon tube with “Sample name
100 μg/mL NaCl 0.1 M.”
l
Weight inside the tube 24 Æ 1 mg of sodium chloride.
l
Transfer in the tube 4 mL of the sample solution
diluted to 100 μg/mL and mix on vortex to dissolve
all the salt.
(c) Free saccharide separation.
l
Initial Equipment preparation.
– Switch on the centrifuge and set the temperature
to 4
C to let it cool.
298
Francesca Micoli et al.