3. Add 10 μL of TEMED into the acrylamide mixture, immediately mix by swirling and pour the acrylamide gel solution into
the glass plate sandwich. Overlay the top of the acrylamide gel
surface with 1 mL of 100% isopropanol and let stand for 10 min
for gel polymerization.
4. Remove the 100% isopropanol by pouring and use tissue paper
to absorb the excess isopropanol.
5. Prepare a 5% stacking gel by mixing 1.446 mL of water, 250 μL
of 40% acrylamide, 254 μL of 1 M Tris–HCl pH 6.8, 20 μL of
10% SDS, and 30 μL of 10% APS in a 25 mL glass beaker and
mix by swirling.
6. Add 5 μL of TEMED into the acrylamide mixture and immediately mix by swirling. Pour the acrylamide gel solution on top
of the separating gel in the glass plate sandwich, insert the
comb to the gel, and then let the gel polymerize for 10 min.
7. Remove the glass plate sandwich from casting component and
assemble in the core electrophoresis apparatus. Add 1Â running buffer to the top and bottom reservoirs and make sure
that the bottom of the gel is submerged in the running buffer
and there is no buffer leakage.
8. To prepare the samples for protein loading, mix 44 μL of the
reserved filtered Zika VLP in Opti-MEM
® media or media
from the mock transfection (see Subheading 3.3, step 1) with
11 μL of 5Â nonreducing sample buffer. Boil samples for 5 min
and immediately place on ice before a short spin down and load
50 μL into each well of the acrylamide gel. Load 4 μL of
molecular weight protein marker into one well as a reference
for protein size.
9. Assemble the lid and power supply to the gel chamber. Run the
gel at a constant 100 V until the marker dye front (bromophenol blue) has reached the end of the gel (approximately 2 h).
10. Turn off the power and remove the acrylamide gel from the
running chamber and transfer the separated protein on to
nitrocellulose membrane by blotting. Soak all components
required for transfer in 1Â transfer buffer before assembly of
the blotting cassette. Place the transfer system fiber pad onto
the dark side of the blotting cassette followed by two layers of
chromatography paper, the acrylamide gel, a 0.22 μm nitrocellulose membrane, two layers of chromatography paper and a
fiber pad, respectively. All components should be soaked in 1Â
transfer buffer before assembly of the transfer sandwich. Place
the blotting cassette into the protein blotting transfer chamber
and then pour 1Â transfer buffer to cover the blotting cassette.
Assemble the lid and power supply and transfer with a constant
300 mA for 1 h.
Zika Virus-Like Particles
199
the glass plate sandwich. Overlay the top of the acrylamide gel
surface with 1 mL of 100% isopropanol and let stand for 10 min
for gel polymerization.
4. Remove the 100% isopropanol by pouring and use tissue paper
to absorb the excess isopropanol.
5. Prepare a 5% stacking gel by mixing 1.446 mL of water, 250 μL
of 40% acrylamide, 254 μL of 1 M Tris–HCl pH 6.8, 20 μL of
10% SDS, and 30 μL of 10% APS in a 25 mL glass beaker and
mix by swirling.
6. Add 5 μL of TEMED into the acrylamide mixture and immediately mix by swirling. Pour the acrylamide gel solution on top
of the separating gel in the glass plate sandwich, insert the
comb to the gel, and then let the gel polymerize for 10 min.
7. Remove the glass plate sandwich from casting component and
assemble in the core electrophoresis apparatus. Add 1Â running buffer to the top and bottom reservoirs and make sure
that the bottom of the gel is submerged in the running buffer
and there is no buffer leakage.
8. To prepare the samples for protein loading, mix 44 μL of the
reserved filtered Zika VLP in Opti-MEM
® media or media
from the mock transfection (see Subheading 3.3, step 1) with
11 μL of 5Â nonreducing sample buffer. Boil samples for 5 min
and immediately place on ice before a short spin down and load
50 μL into each well of the acrylamide gel. Load 4 μL of
molecular weight protein marker into one well as a reference
for protein size.
9. Assemble the lid and power supply to the gel chamber. Run the
gel at a constant 100 V until the marker dye front (bromophenol blue) has reached the end of the gel (approximately 2 h).
10. Turn off the power and remove the acrylamide gel from the
running chamber and transfer the separated protein on to
nitrocellulose membrane by blotting. Soak all components
required for transfer in 1Â transfer buffer before assembly of
the blotting cassette. Place the transfer system fiber pad onto
the dark side of the blotting cassette followed by two layers of
chromatography paper, the acrylamide gel, a 0.22 μm nitrocellulose membrane, two layers of chromatography paper and a
fiber pad, respectively. All components should be soaked in 1Â
transfer buffer before assembly of the transfer sandwich. Place
the blotting cassette into the protein blotting transfer chamber
and then pour 1Â transfer buffer to cover the blotting cassette.
Assemble the lid and power supply and transfer with a constant
300 mA for 1 h.
Zika Virus-Like Particles
199