2.4 Zika VLP
Purification by
Discontinuous Sucrose
Gradient
Centrifugation
1. 0.22 μm PES membrane filter.
2. 100 kDa cutoff column.
3. 1 M Tris–HCl pH 7.5. Weigh 12.11 g of Tris base into a glass
beaker and dissolve with 60 mL of deionized water using a
magnetic stirrer. After the Tris base has dissolved completely,
adjust the pH to 7.5 with concentrated HCl (see Note 8).
Transfer the solution into a measuring cylinder and adjust the
volume to 100 mL with deionized water and then mix thoroughly. The solution can be kept at room temperature.
4. 2.5 M NaCl. Weigh 29.22 g of NaCl into a glass beaker and
dissolve with 150 mL of deionized water using a magnetic
stirrer. After the chemical has completely dissolved, transfer
the solution into a measuring cylinder and adjust the volume
to 200 mL with deionized water and then mix thoroughly. The
solution can be kept at room temperature.
5. 0.5 M EDTA. Weigh 186.12 g of EDTA ∙ Na 2 ∙ 2H 2 O into a
glass beaker and dissolve with 800 mL of deionized water.
Adjust pH to 8.0 with sodium hydroxide (NaOH) (see Note
12). After the chemical has dissolved completely, transfer the
solution into a measuring cylinder and adjust the final volume
to 1 L with deionized water and then sterilize by autoclaving.
The solution can be kept at room temperature.
6. TNE buffer: 10 mM Tris–HCl, 140 mM NaCl, 1 mM EDTA.
Measure 10 mL of 1 M Tris–HCl pH 7.5 solution, 56 mL of
2.5 M NaCl solution and 2 mL of 0.5 M EDTA solution into a
measuring cylinder. Adjust the final volume to 1 L with deionized water and then sterilize by autoclaving. The solution
should be kept at 4
C.
7. 10% sucrose (w/v). Weigh 10 g of sucrose into a glass beaker
and dissolve in 80 mL of TNE buffer using a magnetic stirrer.
After the sucrose has dissolved completely, transfer the solution
into a measuring cylinder and adjust the final volume to
100 mL with TNE buffer and then sterilize by autoclaving.
The solution should be kept at 4
C.
8. 30% sucrose (w/v). Weigh 30 g of sucrose into a glass beaker
and dissolve in 80 mL of TNE buffer using a magnetic stirrer.
After the sucrose has completely dissolved, transfer the solution into a measuring cylinder and adjust the final volume to
100 mL with TNE buffer, then sterilize by autoclaving. The
solution should be kept at 4
C.
9. 60% sucrose (w/v). Weigh 60 g of sucrose into a glass beaker
and dissolve in 60 mL of TNE buffer using a magnetic stirrer.
After the sucrose has dissolved completely, transfer the solution
into a measuring cylinder and adjust the final volume to
100 mL with TNE buffer, then sterilize by autoclaving. The
solution should be kept at 4
C.
190
Atichat Kuadkitkan et al.
Purification by
Discontinuous Sucrose
Gradient
Centrifugation
1. 0.22 μm PES membrane filter.
2. 100 kDa cutoff column.
3. 1 M Tris–HCl pH 7.5. Weigh 12.11 g of Tris base into a glass
beaker and dissolve with 60 mL of deionized water using a
magnetic stirrer. After the Tris base has dissolved completely,
adjust the pH to 7.5 with concentrated HCl (see Note 8).
Transfer the solution into a measuring cylinder and adjust the
volume to 100 mL with deionized water and then mix thoroughly. The solution can be kept at room temperature.
4. 2.5 M NaCl. Weigh 29.22 g of NaCl into a glass beaker and
dissolve with 150 mL of deionized water using a magnetic
stirrer. After the chemical has completely dissolved, transfer
the solution into a measuring cylinder and adjust the volume
to 200 mL with deionized water and then mix thoroughly. The
solution can be kept at room temperature.
5. 0.5 M EDTA. Weigh 186.12 g of EDTA ∙ Na 2 ∙ 2H 2 O into a
glass beaker and dissolve with 800 mL of deionized water.
Adjust pH to 8.0 with sodium hydroxide (NaOH) (see Note
12). After the chemical has dissolved completely, transfer the
solution into a measuring cylinder and adjust the final volume
to 1 L with deionized water and then sterilize by autoclaving.
The solution can be kept at room temperature.
6. TNE buffer: 10 mM Tris–HCl, 140 mM NaCl, 1 mM EDTA.
Measure 10 mL of 1 M Tris–HCl pH 7.5 solution, 56 mL of
2.5 M NaCl solution and 2 mL of 0.5 M EDTA solution into a
measuring cylinder. Adjust the final volume to 1 L with deionized water and then sterilize by autoclaving. The solution
should be kept at 4
C.
7. 10% sucrose (w/v). Weigh 10 g of sucrose into a glass beaker
and dissolve in 80 mL of TNE buffer using a magnetic stirrer.
After the sucrose has dissolved completely, transfer the solution
into a measuring cylinder and adjust the final volume to
100 mL with TNE buffer and then sterilize by autoclaving.
The solution should be kept at 4
C.
8. 30% sucrose (w/v). Weigh 30 g of sucrose into a glass beaker
and dissolve in 80 mL of TNE buffer using a magnetic stirrer.
After the sucrose has completely dissolved, transfer the solution into a measuring cylinder and adjust the final volume to
100 mL with TNE buffer, then sterilize by autoclaving. The
solution should be kept at 4
C.
9. 60% sucrose (w/v). Weigh 60 g of sucrose into a glass beaker
and dissolve in 60 mL of TNE buffer using a magnetic stirrer.
After the sucrose has dissolved completely, transfer the solution
into a measuring cylinder and adjust the final volume to
100 mL with TNE buffer, then sterilize by autoclaving. The
solution should be kept at 4
C.
190
Atichat Kuadkitkan et al.