3.5.2 SDS-PAGE
and Electro-Transfer of Gel
1. Prepare resolving gel solution for 8% SDS-PAGE.
2. Cast gels using an appropriate gel-casting apparatus.
3. Prepare stacking gel solution.
4. Cast stacking gel on top of the 8% resolving gel. Use appropriate comb depending on the number of samples to be analyzed.
5. Prepare samples by mixing 30 μL of cleared protein lysate
(from Subheading 3.5.1) with 6 μL of 6Â SDS sample buffer.
6. Heat samples at 95
C for 5 min.
7. Place gels into an electrophoresis chamber. Fill the appropriate
amount of SDS-PAGE running buffer into the chamber. Load
samples immediately onto the gel. Load pre-stained protein
marker along the samples as well.
8. Run gel at 100 V constant voltage until bromophenol blue
reaches the bottom of the gel.
9. Blot the gel onto nitrocellulose membrane using an appropriate electro-blotting apparatus. Transfer is performed in Towbin
buffer at 300 mA constant power in the cold room (at ~ 4
C)
for 2 h.
10. Stain nitrocellulose membrane with Ponceau S solution to
check protein transfer efficiency. If transfer is satisfactory proceed with immunological detection. If inefficient transfer is
detected (usually missing bands in the high-molecular-weight
range) repeat gel running and blot gel for a longer period of
time (up to 3–4 h).
3.5.3 Immunological
Detection, Quantitation
1. Block membrane in blocking solution with gentle agitation on
an orbital shaker for 1 h at room temperature.
2. To detect GFP and GFP-Renilla luciferase fusion protein,
incubate membrane in diluted polyclonal anti-GFP antibody
(1:2000 dilution in blocking solution) with constant agitation
for 1 h at room temperature.
3. Wash membrane in TBS-T, three times for 10 min with
agitation.
4. Incubate membrane with HRP-conjugated anti-rabbit IgG
(1:10,000 dilution in blocking solution) with constant agitation for 1 h at room temperature.
5. Wash membrane in TBS-T, three times for 10 min with
agitation.
6. Develop membrane using Clarity Western ECL substrate
according to manufacturers’ instructions.
7. Use luminescence gel imaging equipment to image membrane.
The sensor-encoded GFP-Renilla luciferase fusion protein can
be detected on the membrane at ~66 kDa while the miRNA
An In Vivo Reporter System for RNA Silencing
445
and Electro-Transfer of Gel
1. Prepare resolving gel solution for 8% SDS-PAGE.
2. Cast gels using an appropriate gel-casting apparatus.
3. Prepare stacking gel solution.
4. Cast stacking gel on top of the 8% resolving gel. Use appropriate comb depending on the number of samples to be analyzed.
5. Prepare samples by mixing 30 μL of cleared protein lysate
(from Subheading 3.5.1) with 6 μL of 6Â SDS sample buffer.
6. Heat samples at 95
C for 5 min.
7. Place gels into an electrophoresis chamber. Fill the appropriate
amount of SDS-PAGE running buffer into the chamber. Load
samples immediately onto the gel. Load pre-stained protein
marker along the samples as well.
8. Run gel at 100 V constant voltage until bromophenol blue
reaches the bottom of the gel.
9. Blot the gel onto nitrocellulose membrane using an appropriate electro-blotting apparatus. Transfer is performed in Towbin
buffer at 300 mA constant power in the cold room (at ~ 4
C)
for 2 h.
10. Stain nitrocellulose membrane with Ponceau S solution to
check protein transfer efficiency. If transfer is satisfactory proceed with immunological detection. If inefficient transfer is
detected (usually missing bands in the high-molecular-weight
range) repeat gel running and blot gel for a longer period of
time (up to 3–4 h).
3.5.3 Immunological
Detection, Quantitation
1. Block membrane in blocking solution with gentle agitation on
an orbital shaker for 1 h at room temperature.
2. To detect GFP and GFP-Renilla luciferase fusion protein,
incubate membrane in diluted polyclonal anti-GFP antibody
(1:2000 dilution in blocking solution) with constant agitation
for 1 h at room temperature.
3. Wash membrane in TBS-T, three times for 10 min with
agitation.
4. Incubate membrane with HRP-conjugated anti-rabbit IgG
(1:10,000 dilution in blocking solution) with constant agitation for 1 h at room temperature.
5. Wash membrane in TBS-T, three times for 10 min with
agitation.
6. Develop membrane using Clarity Western ECL substrate
according to manufacturers’ instructions.
7. Use luminescence gel imaging equipment to image membrane.
The sensor-encoded GFP-Renilla luciferase fusion protein can
be detected on the membrane at ~66 kDa while the miRNA
An In Vivo Reporter System for RNA Silencing
445