(b) Pyrene butyric acid (PBA) 2.5 mM.
l
Weigh about 7.21 mg of PBA and transfer it in a tube.
l
Calculate the volume of dimethyl sulfoxide (DMSO)
required to dissolve PBA with the following equation:
mL of DMSO ¼ PBA weight (mg)/0.721.
(Where 0.721 ¼ 2.5 mM Â 288.34 (PBA MW)/
1000)
Add the calculated volume of DMSO into the
tube to dissolve PBA.
l
Remark: measure DMSO volume only with pipettes
equipped with polypropylene tips.
(c) Pyridine 20% solution in DMSO (mix in a tube
0.348 mL of pyridine and 1.39 mL of DMSO).
l
Remark: measure DMSO and pyridine volumes only
with pipettes equipped with polypropylene tips.
(d) Adipic acid dihydrazide (ADH) 10 mg/mL standard
solution (150 mg of ADH dissolved in 15 mL of water).
(e) ADH 50 μg/mL standard solution (dilute 100 μL of
ADH 10 mg/mL standard solution by adding 19.9 mL
of water).
(f) ADH 2 μg/mL standard solution (dilute 1000 μL of
ADH 50 μg/mL standard solution by adding 24.0 mL
water).
(g) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) 2 M solution in water.
l
Weigh 668 mg of EDC, transfer it in a 15 mL tube,
and store the tube at À20
C. When the EDC solution will be needed to derivatize the samples add
1742 μL of water and dissolve by vortexing to obtain
the solution.
l
Remark: EDC powder must be dissolved immediately
before its use.
(h) 65% ACN solution (add 26 mL of ACN to 14 mL of
water).
10. Free saccharide analysis.
(a) Deoxycholate sodium salt 1% solution.
l
Weigh 1000 Æ 25 mg of deoxycholate sodium salt and
transfer the powder in a 250 mL glass bottle.
l
With the graduated cylinder add to the bottle a total
amount of 100 mL of water.
l
Place in the bottle the magnetic stirring bar and
homogenize all the content by mixing on a magnetic
stirrer.
O-Antigen Purification and Conjugation
275
l
Weigh about 7.21 mg of PBA and transfer it in a tube.
l
Calculate the volume of dimethyl sulfoxide (DMSO)
required to dissolve PBA with the following equation:
mL of DMSO ¼ PBA weight (mg)/0.721.
(Where 0.721 ¼ 2.5 mM Â 288.34 (PBA MW)/
1000)
Add the calculated volume of DMSO into the
tube to dissolve PBA.
l
Remark: measure DMSO volume only with pipettes
equipped with polypropylene tips.
(c) Pyridine 20% solution in DMSO (mix in a tube
0.348 mL of pyridine and 1.39 mL of DMSO).
l
Remark: measure DMSO and pyridine volumes only
with pipettes equipped with polypropylene tips.
(d) Adipic acid dihydrazide (ADH) 10 mg/mL standard
solution (150 mg of ADH dissolved in 15 mL of water).
(e) ADH 50 μg/mL standard solution (dilute 100 μL of
ADH 10 mg/mL standard solution by adding 19.9 mL
of water).
(f) ADH 2 μg/mL standard solution (dilute 1000 μL of
ADH 50 μg/mL standard solution by adding 24.0 mL
water).
(g) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) 2 M solution in water.
l
Weigh 668 mg of EDC, transfer it in a 15 mL tube,
and store the tube at À20
C. When the EDC solution will be needed to derivatize the samples add
1742 μL of water and dissolve by vortexing to obtain
the solution.
l
Remark: EDC powder must be dissolved immediately
before its use.
(h) 65% ACN solution (add 26 mL of ACN to 14 mL of
water).
10. Free saccharide analysis.
(a) Deoxycholate sodium salt 1% solution.
l
Weigh 1000 Æ 25 mg of deoxycholate sodium salt and
transfer the powder in a 250 mL glass bottle.
l
With the graduated cylinder add to the bottle a total
amount of 100 mL of water.
l
Place in the bottle the magnetic stirring bar and
homogenize all the content by mixing on a magnetic
stirrer.
O-Antigen Purification and Conjugation
275