20. The column oven is heated to decrease the viscosity of the
mobile phases and thus the backpressure of the column. This
enables higher flow rates, shorter run times and it results in
narrower peaks with higher separation efficiencies. Since the
column temperature finally influences the retention time of the
analyzed eluting components, make sure column temperature
is constant at all times. A non-preheated column oven and
varying temperatures during liquid chromatography result in
inconsistent elution times and thus inconclusive chromatograms. The CarboPac™ PA-100 column 2 Â 250mm + guard
column (Dionex™) used in this protocol generates a backpressure of 200 bar at a flow rate of 0.6 mL/min.
21. Cooling is required because the high voltage applied during
electrophoresis generates considerable heat that might lead to
degradation of the sample. Since the analyzed polymers are
negatively charged, make sure that they migrate toward the
positive electrode. The runtime depends on the application.
The electrophoretic mobility of a polymer is dependent on its
physical and chemical properties (e.g., size, charge density) and
should be determined experimentally. The gel shown in Fig. 7
was run for approx. 2 h and was stopped when bromophenol
blue reached the bottom of the gel.
22. The sucrose solution is very viscous and hence difficult to
pipette. The loading buffer is colorless. To minimize mistakes
during loading, mark slots with a permanent marker on the gel
cassette. Cautiously load the samples to avoid contaminating
neighboring wells. It might be beneficial to only fill 50–75% of
the wells and to only load every second well.
Acknowledgements
This study was funded by Deutsche Forschungsgemeinschaft
(DFG, German Research Foundation)—project number
412824531.
References
1. Willis LM, Whitfield C (2013) Structure, biosynthesis, and function of bacterial capsular
polysaccharides
synthesized
by
ABC
transporter-dependent pathways. Carbohydr
Res 378:35–44. https://doi.org/10.1016/j.
carres.2013.05.007
2. Vella M, Pace D (2015) Glycoconjugate vaccines: an update. Expert Opin Biol Ther
15:529–546.
https://doi.org/10.1517/
14712598.2015.993375
3. Berti F, Adamo R (2013) Recent mechanistic
insights on glycoconjugate vaccines and future
perspectives. ACS Chem Biol 8:1653–1663.
https://doi.org/10.1021/cb400423g
4. Costantino P, Rappuoli R, Berti F (2011) The
design of semi-synthetic and synthetic glycoconjugate vaccines. Expert Opin Drug Discov
6:1045–1066.
https://doi.org/10.1517/
17460441.2011.609554
328
Christa Litschko et al.
mobile phases and thus the backpressure of the column. This
enables higher flow rates, shorter run times and it results in
narrower peaks with higher separation efficiencies. Since the
column temperature finally influences the retention time of the
analyzed eluting components, make sure column temperature
is constant at all times. A non-preheated column oven and
varying temperatures during liquid chromatography result in
inconsistent elution times and thus inconclusive chromatograms. The CarboPac™ PA-100 column 2 Â 250mm + guard
column (Dionex™) used in this protocol generates a backpressure of 200 bar at a flow rate of 0.6 mL/min.
21. Cooling is required because the high voltage applied during
electrophoresis generates considerable heat that might lead to
degradation of the sample. Since the analyzed polymers are
negatively charged, make sure that they migrate toward the
positive electrode. The runtime depends on the application.
The electrophoretic mobility of a polymer is dependent on its
physical and chemical properties (e.g., size, charge density) and
should be determined experimentally. The gel shown in Fig. 7
was run for approx. 2 h and was stopped when bromophenol
blue reached the bottom of the gel.
22. The sucrose solution is very viscous and hence difficult to
pipette. The loading buffer is colorless. To minimize mistakes
during loading, mark slots with a permanent marker on the gel
cassette. Cautiously load the samples to avoid contaminating
neighboring wells. It might be beneficial to only fill 50–75% of
the wells and to only load every second well.
Acknowledgements
This study was funded by Deutsche Forschungsgemeinschaft
(DFG, German Research Foundation)—project number
412824531.
References
1. Willis LM, Whitfield C (2013) Structure, biosynthesis, and function of bacterial capsular
polysaccharides
synthesized
by
ABC
transporter-dependent pathways. Carbohydr
Res 378:35–44. https://doi.org/10.1016/j.
carres.2013.05.007
2. Vella M, Pace D (2015) Glycoconjugate vaccines: an update. Expert Opin Biol Ther
15:529–546.
https://doi.org/10.1517/
14712598.2015.993375
3. Berti F, Adamo R (2013) Recent mechanistic
insights on glycoconjugate vaccines and future
perspectives. ACS Chem Biol 8:1653–1663.
https://doi.org/10.1021/cb400423g
4. Costantino P, Rappuoli R, Berti F (2011) The
design of semi-synthetic and synthetic glycoconjugate vaccines. Expert Opin Drug Discov
6:1045–1066.
https://doi.org/10.1517/
17460441.2011.609554
328
Christa Litschko et al.