3. Cast a 12% SDS-PAGE gel under a fume hood. To do so,
prepare the resolving gel mix (10 mL of 30% acrylamide/bisacrylamide 37.5/1, 8.3 mL of resolving buffer, and 6.7 mL of
water—enough for 3 gels if using the standard 7 Â 10 cm
BioRad plates with spacers for 1.5 mm thickness). Mix well,
then add 250 μL of 10% APS, mix well again, add 25 μL
TEMED, and briefly mix once more. Quickly pour/pipette
the liquid between the glass plates in the gel-casting system
up to 2/3 or ¾ of the height. Rapidly overlay the solution with
500 μL ethanol to flatten its surface and seal it from air. Let the
solution polymerize for 15 min, then pour out the ethanol, and
carefully dry the top of the gel with blotting paper. Prepare the
stacking gel mix (2 mL of 30% acrylamide/bis-acrylamide
37.5/1, 3 mL of stacking buffer, and 10 mL of water). Mix
well, add 150 μL of 10% APS, mix well again, add 15 μL
TEMED, and briefly mix once more. Quickly pour/pipette
the liquid between the glass plates on the resolving gel until it
is filled up to the top and insert the comb. Allow gel polymerization for 10–15 min at room temperature.
4. Place the glass plates containing the gel into the running tank
filled with migration buffer and carefully remove the comb.
Load the samples (10 μL each) and the protein size marker
(5 μL) into the different wells of the gel, and then perform the
electrophoresis at 80 V until the proteins reach the resolving
part of the gel. Increase the voltage to 120–150 V and continue
electrophoresis until the 25–30 kDa protein of the size marker
has migrated into the lower third of the gel. Open the gel
container to remove the gel.
5. Immerse the PVDF membrane, previously cut to the size of the
gel, in 80% ethanol for 5 min. Imbibe two gel-sized pieces of
blotting paper in transfer buffer. Assemble the transfer “sandwich” provided with the electro-transfer system: open the
holder and place sponge, blotting paper, gel, and membrane
in this order. Carefully remove all bubbles and then place the
other blotting paper and sponge on top. Close the sandwich
holder and place it into the transfer tank (with the gel oriented
toward the cathode and the membrane oriented toward the
anode) along with the provided ice container. Connect to the
power supply and allow proteins to migrate from the gel onto
the membrane at 4
C for 90 min at 80 V. Stir the buffer by
placing a stir bar into the tank and the tank onto a magnetic
stirrer in order to homogenize the buffer composition and
temperature during transfer.
6. Remove the membrane from transfer apparatus, place it into a
crystal box containing PBS/Tween, and allow gentle oscillation for 5 min.
Pull-Down of Viral Double-Stranded RNA
321
prepare the resolving gel mix (10 mL of 30% acrylamide/bisacrylamide 37.5/1, 8.3 mL of resolving buffer, and 6.7 mL of
water—enough for 3 gels if using the standard 7 Â 10 cm
BioRad plates with spacers for 1.5 mm thickness). Mix well,
then add 250 μL of 10% APS, mix well again, add 25 μL
TEMED, and briefly mix once more. Quickly pour/pipette
the liquid between the glass plates in the gel-casting system
up to 2/3 or ¾ of the height. Rapidly overlay the solution with
500 μL ethanol to flatten its surface and seal it from air. Let the
solution polymerize for 15 min, then pour out the ethanol, and
carefully dry the top of the gel with blotting paper. Prepare the
stacking gel mix (2 mL of 30% acrylamide/bis-acrylamide
37.5/1, 3 mL of stacking buffer, and 10 mL of water). Mix
well, add 150 μL of 10% APS, mix well again, add 15 μL
TEMED, and briefly mix once more. Quickly pour/pipette
the liquid between the glass plates on the resolving gel until it
is filled up to the top and insert the comb. Allow gel polymerization for 10–15 min at room temperature.
4. Place the glass plates containing the gel into the running tank
filled with migration buffer and carefully remove the comb.
Load the samples (10 μL each) and the protein size marker
(5 μL) into the different wells of the gel, and then perform the
electrophoresis at 80 V until the proteins reach the resolving
part of the gel. Increase the voltage to 120–150 V and continue
electrophoresis until the 25–30 kDa protein of the size marker
has migrated into the lower third of the gel. Open the gel
container to remove the gel.
5. Immerse the PVDF membrane, previously cut to the size of the
gel, in 80% ethanol for 5 min. Imbibe two gel-sized pieces of
blotting paper in transfer buffer. Assemble the transfer “sandwich” provided with the electro-transfer system: open the
holder and place sponge, blotting paper, gel, and membrane
in this order. Carefully remove all bubbles and then place the
other blotting paper and sponge on top. Close the sandwich
holder and place it into the transfer tank (with the gel oriented
toward the cathode and the membrane oriented toward the
anode) along with the provided ice container. Connect to the
power supply and allow proteins to migrate from the gel onto
the membrane at 4
C for 90 min at 80 V. Stir the buffer by
placing a stir bar into the tank and the tank onto a magnetic
stirrer in order to homogenize the buffer composition and
temperature during transfer.
6. Remove the membrane from transfer apparatus, place it into a
crystal box containing PBS/Tween, and allow gentle oscillation for 5 min.
Pull-Down of Viral Double-Stranded RNA
321