Prepare all solutions with ultrapure water and analytical grade
reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Solutions containing volatile solvents
need to be prepared in a fume hood. Follow all waste disposal
regulations. The method requires a gel chamber that enables the
constant cooling of the system during electrophoresis and that
allows for the installation of gel cassettes of sufficient size (see
Note 10). Electrophoresis was performed using the vertical protein
electrophoresis chamber from Biometra with an included built-in
water-cooling system used in combination with the minichiller
from Huber.
1. 1Â TBE (100 mM Tris, 100 mM boric acid, 2.5 mM EDTA).
For 1 L of a 10Â TBE stock solution (1 M Tris, 1 M boric acid,
25 mM EDTA), weigh 121.14 g of Tris, 61.85 g of boric acid,
and 9.3 g of EDTA in a 1 L volumetric flask. Add about
600 mL of water and dissolve the salts. Subsequently add
water to a volume of 1 L and autoclave the buffer. For 1Â
TBE, dilute 10Â TBE 1:10 with water.
2. 40% acrylamide solution (commercially available). Store at
4
C.
3. 2% bisacrylamide (commercially available). Store at 4
C.
4. 10% ammonium persulfate (APS): Weigh 1 g APS in a 10 mL
volumetric flask and add water to the graduation mark. Store at
4
C.
5. N,N,N
0 ,N
0 -tetramethylethylenediamine (TEMED). Store at
4
C.
6. Loading buffer: 2 M sucrose in 10Â TBE. To obtain 50 mL,
dissolve 34.23 g of sucrose in 10Â TBE.
7. Polysaccharide marker (dissolved 1:2 in loading buffer): 0.05%
trypan blue (corresponds to polySia-DP100), 0.02% xylene
cyanol (corresponds to polySia-DP52), 0.02% bromophenol
blue (corresponds to polySia-DP19), 0.02% bromo cresol purple (corresponds to polySia-DP11), 0.02% phenol red (corresponds to polySia-DP6) (see Note 11).
8. Fixing solution: 40% ethanol, 5% acetic acid. For 100 mL of
fixing solution, mix 40 mL of ethanol and 5 mL of acetic acid
and add water to a volume of 100 mL. Should be prepared
freshly for each use (see Note 12).
9. Alcian blue solution: 0.5% alcian blue, 3% acetic acid. Weigh
500 mg of alcian blue, add 3 mL of acetic acid, and add water
to a volume of 100 mL. Filter the solution. The staining
solution can be reused at least five to ten times (see Note 12).
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
319
reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Solutions containing volatile solvents
need to be prepared in a fume hood. Follow all waste disposal
regulations. The method requires a gel chamber that enables the
constant cooling of the system during electrophoresis and that
allows for the installation of gel cassettes of sufficient size (see
Note 10). Electrophoresis was performed using the vertical protein
electrophoresis chamber from Biometra with an included built-in
water-cooling system used in combination with the minichiller
from Huber.
1. 1Â TBE (100 mM Tris, 100 mM boric acid, 2.5 mM EDTA).
For 1 L of a 10Â TBE stock solution (1 M Tris, 1 M boric acid,
25 mM EDTA), weigh 121.14 g of Tris, 61.85 g of boric acid,
and 9.3 g of EDTA in a 1 L volumetric flask. Add about
600 mL of water and dissolve the salts. Subsequently add
water to a volume of 1 L and autoclave the buffer. For 1Â
TBE, dilute 10Â TBE 1:10 with water.
2. 40% acrylamide solution (commercially available). Store at
4
C.
3. 2% bisacrylamide (commercially available). Store at 4
C.
4. 10% ammonium persulfate (APS): Weigh 1 g APS in a 10 mL
volumetric flask and add water to the graduation mark. Store at
4
C.
5. N,N,N
0 ,N
0 -tetramethylethylenediamine (TEMED). Store at
4
C.
6. Loading buffer: 2 M sucrose in 10Â TBE. To obtain 50 mL,
dissolve 34.23 g of sucrose in 10Â TBE.
7. Polysaccharide marker (dissolved 1:2 in loading buffer): 0.05%
trypan blue (corresponds to polySia-DP100), 0.02% xylene
cyanol (corresponds to polySia-DP52), 0.02% bromophenol
blue (corresponds to polySia-DP19), 0.02% bromo cresol purple (corresponds to polySia-DP11), 0.02% phenol red (corresponds to polySia-DP6) (see Note 11).
8. Fixing solution: 40% ethanol, 5% acetic acid. For 100 mL of
fixing solution, mix 40 mL of ethanol and 5 mL of acetic acid
and add water to a volume of 100 mL. Should be prepared
freshly for each use (see Note 12).
9. Alcian blue solution: 0.5% alcian blue, 3% acetic acid. Weigh
500 mg of alcian blue, add 3 mL of acetic acid, and add water
to a volume of 100 mL. Filter the solution. The staining
solution can be reused at least five to ten times (see Note 12).
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
319