3.3 Gene Silencing
Evaluation
The basic approach for assessing gene silencing in our laboratory is
performing immunoblotting of the recovered recombinant
L. biflexa cells. This strategy is based on the availability of antiserum
against the protein encoded by the gene.
1. Collect the recombinant L. biflexa cells grown in liquid EMJH
medium by centrifugation (4000 Â g, room temperature,
15 min). Wash the bacterial cell pellet twice with PBS (see
Note 8).
2. Resuspend the cells in 100 μL of PBS and then check the
OD 420 of the samples, using PBS as the blank. After checking
the densitometry reading, dilute all samples with PBS to the
same OD, for normalization purposes. We use a final OD 420 of
5–10. If your target protein is poorly expressed, we recommend using a higher cell density (see Note 9).
3. Add 25 μL of 5Â SDS-PAGE buffer to the 100 μL of bacterial
solutions, vortex briefly, and boil the normalized samples at
96
C for 10 min.
4. Subject the samples to SDS-PAGE (percentage depending on
the molecular weight of the target protein). For protein ranging from 20 to 60 kDa, we use 12% gels. For preparation of the
separating gel, mix 4 mL of 30% polyacrylamide mixture,
3.3 mL of water, and 2.5 mL of 1.5 M Tris, pH 8.8, in a
50 mL tube. Add 100 μL of 10% SDS, 100 μL of 10% ammonium persulfate, and 10 μL of TEMED, mix gently, and pour
gel in a 7.25 cm  10 cm  1-mm gel cassette. Normally, 7 mL
is sufficient for the resolving gel. Overlay the gel with isopropanol or water, saving around 3 cm for the stacking gel.
5. For preparation of the stacking gel, mix 0.83 mL of 30%
acrylamide mixture, 3.4 mL water, and 0.63 mL 1 M Tris,
pH 6.8, in a 50 mL tube. Add 50 μL of 10% SDS, 50 μL of
10% ammonium persulfate, and 5 μL of TEMED. Insert the gel
comb immediately, avoiding the formation of bubbles.
6. For the electrophoresis, include protein standards. Set the
amperage at 25 mA per gel, and run the samples until the dye
reaches the bottom of the gel.
7. As the electrophoresis is ending, prepare the system for protein
transfer. Immerse three to four sheets of filter paper in 1Â
transfer buffer in a plastic container, then the nitrocellulose
membrane, combining the elements on the surface of the
semidry transfer system. When the run is over, carefully take
the gel from the electrophoresis cassette, and let it sit in the
transfer buffer for about 5 min. Next, place the gel over the
nitrocellulose membrane and then cover it with three to four
more sheets of filter paper, previously soaked in transfer buffer.
118
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento
Evaluation
The basic approach for assessing gene silencing in our laboratory is
performing immunoblotting of the recovered recombinant
L. biflexa cells. This strategy is based on the availability of antiserum
against the protein encoded by the gene.
1. Collect the recombinant L. biflexa cells grown in liquid EMJH
medium by centrifugation (4000 Â g, room temperature,
15 min). Wash the bacterial cell pellet twice with PBS (see
Note 8).
2. Resuspend the cells in 100 μL of PBS and then check the
OD 420 of the samples, using PBS as the blank. After checking
the densitometry reading, dilute all samples with PBS to the
same OD, for normalization purposes. We use a final OD 420 of
5–10. If your target protein is poorly expressed, we recommend using a higher cell density (see Note 9).
3. Add 25 μL of 5Â SDS-PAGE buffer to the 100 μL of bacterial
solutions, vortex briefly, and boil the normalized samples at
96
C for 10 min.
4. Subject the samples to SDS-PAGE (percentage depending on
the molecular weight of the target protein). For protein ranging from 20 to 60 kDa, we use 12% gels. For preparation of the
separating gel, mix 4 mL of 30% polyacrylamide mixture,
3.3 mL of water, and 2.5 mL of 1.5 M Tris, pH 8.8, in a
50 mL tube. Add 100 μL of 10% SDS, 100 μL of 10% ammonium persulfate, and 10 μL of TEMED, mix gently, and pour
gel in a 7.25 cm  10 cm  1-mm gel cassette. Normally, 7 mL
is sufficient for the resolving gel. Overlay the gel with isopropanol or water, saving around 3 cm for the stacking gel.
5. For preparation of the stacking gel, mix 0.83 mL of 30%
acrylamide mixture, 3.4 mL water, and 0.63 mL 1 M Tris,
pH 6.8, in a 50 mL tube. Add 50 μL of 10% SDS, 50 μL of
10% ammonium persulfate, and 5 μL of TEMED. Insert the gel
comb immediately, avoiding the formation of bubbles.
6. For the electrophoresis, include protein standards. Set the
amperage at 25 mA per gel, and run the samples until the dye
reaches the bottom of the gel.
7. As the electrophoresis is ending, prepare the system for protein
transfer. Immerse three to four sheets of filter paper in 1Â
transfer buffer in a plastic container, then the nitrocellulose
membrane, combining the elements on the surface of the
semidry transfer system. When the run is over, carefully take
the gel from the electrophoresis cassette, and let it sit in the
transfer buffer for about 5 min. Next, place the gel over the
nitrocellulose membrane and then cover it with three to four
more sheets of filter paper, previously soaked in transfer buffer.
118
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento