2.3 Zika VLP
Detection by
Western Blot
1. 1.5 M Tris-HCl pH 8.8. Weigh 27.26 g of Tris base into a glass
beaker and dissolve with 80 mL of deionized water using a
magnetic stirrer until completely dissolved. Adjust the pH to
8.8 using concentrated HCl (see Note 8). Transfer the solution
into a measuring cylinder then add deionized water to make
the final volume of 150 mL and store at room temperature.
2. 1 M Tris–HCl pH 6.8. Weigh 12.1 g of Tris base into a glass
beaker and dissolve with 50 mL of deionized water using a
magnetic stirrer until completely dissolved. Adjust the pH to
6.8 using concentrated HCl (see Note 8). Transfer the solution
into a measuring cylinder then add deionized water to adjust
the final volume to 100 mL and store at room temperature.
3. 10% SDS (w/v). Weigh 10 g of sodium dodecyl sulfate (SDS)
(see Note 9) into a glass beaker and dissolve with deionized
water using a magnetic stirrer. The solution can be heated to
68
C to improve the solubility. After the SDS has dissolved
completely, adjust the volume to 100 mL with deionized water
and mix thoroughly. The solution can be kept at room
temperature.
4. 10% ammonium persulfate (APS) (w/v). Weigh 0.1 g of APS
into a 1.5 mL tube and dissolve with 1 mL of deionized water
by vigorously vortex. The solution should be freshly prepared
and can be kept at 4
C for 1 week.
5. 40% acrylamide–bis solution (29.1:0.9).
6. N,N,N
0 ,N
0 -tetramethyl-ethylenediamine (TEMED).
7. 5Â nonreducing sample buffer: 0.3 M Tris–HCl pH 6.8, 5%
SDS, 50% glycerol, 0.015% bromophenol blue. Weigh 1.5 g of
SDS into a glass beaker and dissolve with 9 mL of 1 M Tris–
HCl pH 6.8 using a magnetic stirrer. The solution can be
heated to 68
C to improve the solubility. Then add 15 mL
of 100% glycerol into the solution and continue mixing. In
parallel, weigh 4.5 mg of bromophenol blue into a 5 mL tube
and dissolve with 2 mL of deionized water by vortexing. After
the solution is completely solubilized, add the bromophenol
blue solution into the Tris–HCl/SDS/glycerol solution and
adjust the volume to 30 mL with deionized water and mix
thoroughly. The solution can be kept at room temperature.
8. 10Â SDS-PAGE running buffer: 0.25 M Tris–HCl pH 8.3,
1.92 M glycine, 1% SDS. Weigh 30.2 g of Tris base, 141.4 of
glycine and 10 g of SDS into a glass beaker and dissolve with
500 mL of deionized water using a magnetic stirrer. The pH of
the buffer should be 8.3, and pH adjustment is not normally
required. After all the chemicals have dissolved completely,
adjust the volume to 1 L with deionized water and mix thoroughly. The solution can be kept at room temperature.
188
Atichat Kuadkitkan et al.
Detection by
Western Blot
1. 1.5 M Tris-HCl pH 8.8. Weigh 27.26 g of Tris base into a glass
beaker and dissolve with 80 mL of deionized water using a
magnetic stirrer until completely dissolved. Adjust the pH to
8.8 using concentrated HCl (see Note 8). Transfer the solution
into a measuring cylinder then add deionized water to make
the final volume of 150 mL and store at room temperature.
2. 1 M Tris–HCl pH 6.8. Weigh 12.1 g of Tris base into a glass
beaker and dissolve with 50 mL of deionized water using a
magnetic stirrer until completely dissolved. Adjust the pH to
6.8 using concentrated HCl (see Note 8). Transfer the solution
into a measuring cylinder then add deionized water to adjust
the final volume to 100 mL and store at room temperature.
3. 10% SDS (w/v). Weigh 10 g of sodium dodecyl sulfate (SDS)
(see Note 9) into a glass beaker and dissolve with deionized
water using a magnetic stirrer. The solution can be heated to
68
C to improve the solubility. After the SDS has dissolved
completely, adjust the volume to 100 mL with deionized water
and mix thoroughly. The solution can be kept at room
temperature.
4. 10% ammonium persulfate (APS) (w/v). Weigh 0.1 g of APS
into a 1.5 mL tube and dissolve with 1 mL of deionized water
by vigorously vortex. The solution should be freshly prepared
and can be kept at 4
C for 1 week.
5. 40% acrylamide–bis solution (29.1:0.9).
6. N,N,N
0 ,N
0 -tetramethyl-ethylenediamine (TEMED).
7. 5Â nonreducing sample buffer: 0.3 M Tris–HCl pH 6.8, 5%
SDS, 50% glycerol, 0.015% bromophenol blue. Weigh 1.5 g of
SDS into a glass beaker and dissolve with 9 mL of 1 M Tris–
HCl pH 6.8 using a magnetic stirrer. The solution can be
heated to 68
C to improve the solubility. Then add 15 mL
of 100% glycerol into the solution and continue mixing. In
parallel, weigh 4.5 mg of bromophenol blue into a 5 mL tube
and dissolve with 2 mL of deionized water by vortexing. After
the solution is completely solubilized, add the bromophenol
blue solution into the Tris–HCl/SDS/glycerol solution and
adjust the volume to 30 mL with deionized water and mix
thoroughly. The solution can be kept at room temperature.
8. 10Â SDS-PAGE running buffer: 0.25 M Tris–HCl pH 8.3,
1.92 M glycine, 1% SDS. Weigh 30.2 g of Tris base, 141.4 of
glycine and 10 g of SDS into a glass beaker and dissolve with
500 mL of deionized water using a magnetic stirrer. The pH of
the buffer should be 8.3, and pH adjustment is not normally
required. After all the chemicals have dissolved completely,
adjust the volume to 1 L with deionized water and mix thoroughly. The solution can be kept at room temperature.
188
Atichat Kuadkitkan et al.