3. Analyze the conjugation mixture by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and HPLCSEC to verify conjugate formation, comparing it with free
protein and free saccharide (Fig. 3).
4. Purify the conjugate by size exclusion chromatography on
16/90 cm Sephacryl S-300 HR column eluting at 0.5 mL/
min in 50 mM phosphate buffer, 150 mM NaCl, pH 7.2 (see
Note 19).
5. Characterize the conjugate by:
(a) micro-BCA for total protein content.
(b) Phenol sulfuric acid assay for quantifying total sugar content and HPAEC-PAD analysis for sugar composition and
content.
(c) OAg to protein w/w ratio.
(d) HPLC-SEC for molecular size distribution.
(e)
1 H NMR for OAg identity and O-acetyl content.
(f) Free saccharide analysis (see Note 20).
l
Conjugate precipitation with deoxycholate for separation from free saccharide.
l
Quantification of free saccharide (e.g., through
HPAEC-PAD).
3.2.3 Analytical Methods
3.3 OAg
and glycoconjugate
characterization
1. Phenol sulfuric assay [15].
(a) Calibration curve preparation.
l
Using water, prepare in labeled tubes, in duplicate, the
dilutions of the Glc standard solution (0.5 mg/mL), as
indicated in Table 1.
l
Label each tube with the corresponding standard
concentration.
(b) Sample preparation.
l
Ensure that sample concentration is in the range of the
calibration curve. If not, dilute it with water (mix samples before dilution in order to homogenize the vial
content).
l
Put 200 μL of each sample dilution in a tube. Label
each tube with the corresponding sample name. Each
sample is analyzed in duplicate.
(c) Sample/Standard treatment.
l
Put the standard and sample tubes in a suitable rack
which must remain within a chemical hood for all the
following procedure.
O-Antigen Purification and Conjugation
281
protein and free saccharide (Fig. 3).
4. Purify the conjugate by size exclusion chromatography on
16/90 cm Sephacryl S-300 HR column eluting at 0.5 mL/
min in 50 mM phosphate buffer, 150 mM NaCl, pH 7.2 (see
Note 19).
5. Characterize the conjugate by:
(a) micro-BCA for total protein content.
(b) Phenol sulfuric acid assay for quantifying total sugar content and HPAEC-PAD analysis for sugar composition and
content.
(c) OAg to protein w/w ratio.
(d) HPLC-SEC for molecular size distribution.
(e)
1 H NMR for OAg identity and O-acetyl content.
(f) Free saccharide analysis (see Note 20).
l
Conjugate precipitation with deoxycholate for separation from free saccharide.
l
Quantification of free saccharide (e.g., through
HPAEC-PAD).
3.2.3 Analytical Methods
3.3 OAg
and glycoconjugate
characterization
1. Phenol sulfuric assay [15].
(a) Calibration curve preparation.
l
Using water, prepare in labeled tubes, in duplicate, the
dilutions of the Glc standard solution (0.5 mg/mL), as
indicated in Table 1.
l
Label each tube with the corresponding standard
concentration.
(b) Sample preparation.
l
Ensure that sample concentration is in the range of the
calibration curve. If not, dilute it with water (mix samples before dilution in order to homogenize the vial
content).
l
Put 200 μL of each sample dilution in a tube. Label
each tube with the corresponding sample name. Each
sample is analyzed in duplicate.
(c) Sample/Standard treatment.
l
Put the standard and sample tubes in a suitable rack
which must remain within a chemical hood for all the
following procedure.
O-Antigen Purification and Conjugation
281