We recommend using a roll to remove any air bubbles from the
“sandwich.”
8. Set the transfer to 0.5 A for 30 min.
9. When finished, carefully remove the membrane and place it in a
new plastic container. For evaluating the transfer efficiency, add
10 mL of Ponceau S solution and incubate the membrane for
5 min. Afterward, recover the solution and rinse the membrane
with distilled water to decrease the background and visualize
the transferred proteins. Mark every lane and standard proteins
with a pencil to interpret the results. Ponceau S solutions can
be reused several times. When loss of staining capacity is
observed, discard the solution appropriately and use a fresh
one. We recommend photographing the transferred protein
to verify the normalization of the samples.
10. Wash the membrane with PBS-T until Ponceau S is removed.
Next, add the blocking buffer (milk or BSA) to the membrane
and incubate for 30 min at 37
C.
11. Discard the blocking buffer and add the primary antibody
solution. Dilute the polyclonal antiserum in blocking buffer,
and incubate for 1 h at room temperature with moderate
shaking (see Notes 10 and 11). We use 10 mL of solution per
membrane. Dilution of the antiserum may vary. We start the
experiments with 1:1000 dilution, and depending on the outcome, we adjust the concentration.
12. Wash the membrane three times with PBS-T and incubate with
secondary antibody solution in blocking buffer. We use horseradish peroxidase-conjugated anti-mouse IgG (1:5000). Incubate at room temperature for 40 min with gentle shaking.
13. Wash the membranes five times with PBS-T and then add the
chemiluminescent substrate for visualization of reactivity. For
gene silencing, the expected results are intense signal in
L. biflexa cells containing pMaOri.dCas9 alone, accompanied
by a slightly reduced signal when dCas9 is expressed along the
sgRNA pairing to the template strand and abolished signal
when the sgRNA is capable of pairing to the coding strand.
This result can be associated with measurable phenotypes, as
described in Note 12. We recommend adding spectinomycin
for maintaining the mutant cultures (see Note 13).
4 Notes
1. Even though our experiments were performed in the saprophyte L. biflexa, it is anticipated that our tool can be applied to
pathogenic strains.
Gene Silencing by Single-Guide RNA and dCas9
119
“sandwich.”
8. Set the transfer to 0.5 A for 30 min.
9. When finished, carefully remove the membrane and place it in a
new plastic container. For evaluating the transfer efficiency, add
10 mL of Ponceau S solution and incubate the membrane for
5 min. Afterward, recover the solution and rinse the membrane
with distilled water to decrease the background and visualize
the transferred proteins. Mark every lane and standard proteins
with a pencil to interpret the results. Ponceau S solutions can
be reused several times. When loss of staining capacity is
observed, discard the solution appropriately and use a fresh
one. We recommend photographing the transferred protein
to verify the normalization of the samples.
10. Wash the membrane with PBS-T until Ponceau S is removed.
Next, add the blocking buffer (milk or BSA) to the membrane
and incubate for 30 min at 37
C.
11. Discard the blocking buffer and add the primary antibody
solution. Dilute the polyclonal antiserum in blocking buffer,
and incubate for 1 h at room temperature with moderate
shaking (see Notes 10 and 11). We use 10 mL of solution per
membrane. Dilution of the antiserum may vary. We start the
experiments with 1:1000 dilution, and depending on the outcome, we adjust the concentration.
12. Wash the membrane three times with PBS-T and incubate with
secondary antibody solution in blocking buffer. We use horseradish peroxidase-conjugated anti-mouse IgG (1:5000). Incubate at room temperature for 40 min with gentle shaking.
13. Wash the membranes five times with PBS-T and then add the
chemiluminescent substrate for visualization of reactivity. For
gene silencing, the expected results are intense signal in
L. biflexa cells containing pMaOri.dCas9 alone, accompanied
by a slightly reduced signal when dCas9 is expressed along the
sgRNA pairing to the template strand and abolished signal
when the sgRNA is capable of pairing to the coding strand.
This result can be associated with measurable phenotypes, as
described in Note 12. We recommend adding spectinomycin
for maintaining the mutant cultures (see Note 13).
4 Notes
1. Even though our experiments were performed in the saprophyte L. biflexa, it is anticipated that our tool can be applied to
pathogenic strains.
Gene Silencing by Single-Guide RNA and dCas9
119