1. Add all components of the reaction mixture to a tube, gently
mix. Start the reaction by adding the capsule polymerase (see
Note 16).
2. Incubate the reaction mixture overnight at 37
C (see Notes 17
and 18).
3. Stop reaction by heat shock at 60
C for 5 min (see Note 19).
4. Centrifuge sample at 20,000 Â g for 2 min to spin down
precipitated protein and potential aggregates prior to analysis
by HPLC.
3.2 HPLC Assay
When using substrate concentrations as shown in Table 1, 3–5 μL
of sample is required for the detection of nucleotides at 280 nm and
40–50 μL for the detection of capsule polymer at 214 nm. The
samples are cooled to 15
C in the autosampler to avoid spontaneous hydrolysis of sample ingredients and evaporation. The column
oven is heated to 50
C and the flow rate is set to 0.6 mL/min.
1. Transfer the supernatant of the centrifuged sample to a tube
suitable for your HPLC device and autosampler. Leave some of
the supernatant in the tube to avoid unnecessary stirring of
precipitated protein. 65 μL of supernatant will suffice to perform the analysis. Cool the autosampler to 15
C. Use tube
seals with precut slits to avoid evaporation of the sample in the
autosampler.
2. Heat the column oven to 50
C (see Note 20).
3. Equilibrate the system and column with mobile phase 1.
4. Start the instrument method for the analysis of nucleotides
with an absorption wavelength of 280 nm. Load a sample
volume of 3–5 μL (see Subheading 2.2, item 5).
5. For the analysis of UV-active polymer, start the instrument
method with an absorption wavelength of 214 nm and load
40–50 μL of sample (see Subheading 2.2, item 6).
6. Analyze the chromatograms. Identify the sample constituents
by comparison with the standards (Figs. 5 and 6).
3.3 Visualization
of Polymer
in a High-Percentage
PAGE
1. Prepare the acrylamide–bisacrylamide solution according to
the table below (Table 2). A separating gel is sufficient for
this method, no stacking gel is needed.
2. After the addition of TEMED, immediately pour the unpolymerized gel solution between the glass plates and insert a comb
without introducing air bubbles.
3. Allow the polymerization of the gel for a minimum of 30 min.
4. Insert the gel cassette into the gel chamber and fill the chamber
with 1Â TBE.
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
321
mix. Start the reaction by adding the capsule polymerase (see
Note 16).
2. Incubate the reaction mixture overnight at 37
C (see Notes 17
and 18).
3. Stop reaction by heat shock at 60
C for 5 min (see Note 19).
4. Centrifuge sample at 20,000 Â g for 2 min to spin down
precipitated protein and potential aggregates prior to analysis
by HPLC.
3.2 HPLC Assay
When using substrate concentrations as shown in Table 1, 3–5 μL
of sample is required for the detection of nucleotides at 280 nm and
40–50 μL for the detection of capsule polymer at 214 nm. The
samples are cooled to 15
C in the autosampler to avoid spontaneous hydrolysis of sample ingredients and evaporation. The column
oven is heated to 50
C and the flow rate is set to 0.6 mL/min.
1. Transfer the supernatant of the centrifuged sample to a tube
suitable for your HPLC device and autosampler. Leave some of
the supernatant in the tube to avoid unnecessary stirring of
precipitated protein. 65 μL of supernatant will suffice to perform the analysis. Cool the autosampler to 15
C. Use tube
seals with precut slits to avoid evaporation of the sample in the
autosampler.
2. Heat the column oven to 50
C (see Note 20).
3. Equilibrate the system and column with mobile phase 1.
4. Start the instrument method for the analysis of nucleotides
with an absorption wavelength of 280 nm. Load a sample
volume of 3–5 μL (see Subheading 2.2, item 5).
5. For the analysis of UV-active polymer, start the instrument
method with an absorption wavelength of 214 nm and load
40–50 μL of sample (see Subheading 2.2, item 6).
6. Analyze the chromatograms. Identify the sample constituents
by comparison with the standards (Figs. 5 and 6).
3.3 Visualization
of Polymer
in a High-Percentage
PAGE
1. Prepare the acrylamide–bisacrylamide solution according to
the table below (Table 2). A separating gel is sufficient for
this method, no stacking gel is needed.
2. After the addition of TEMED, immediately pour the unpolymerized gel solution between the glass plates and insert a comb
without introducing air bubbles.
3. Allow the polymerization of the gel for a minimum of 30 min.
4. Insert the gel cassette into the gel chamber and fill the chamber
with 1Â TBE.
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
321