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3 protocols using strep tactin conjugated to horseradish peroxidase

1

Reagents for RNA-related Experiments

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The following reagents were purchased: mouse mAb J2 (Scicons); Alexa Fluor 488-goat anti-mouse IgG secondary antibody (Molecular Probes, Life Technologies); Phi6 dsRNA (Finnzymes, Thermo Fisher Scientific); dsRNA ladder (New England Biolabs); High MW poly (A:U) and poly (I:C) (InvivoGen); GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); Silencer negative control 1 siRNA (Ambion, Life Technologies); Strep-Tactin conjugated to horseradish peroxidase (IBA Lifesciences); pNPP (Sigma-Aldrich), mMESSAGE mMACHINE T7 transcription kit (Ambion, Life Technologies), Lumi-Light western blotting substrate (Roche Diagnostics).
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2

Quantitative Western Blot Analysis

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Mycoplasma cell lysates were quantified using the Pierce ™ BCA Protein Assay Kit. Cell extracts were separated by electrophoresis using NuPage™ 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and proteins transferred onto nitrocellulose membranes using an iBlot™ dry system (Invitrogen). Membranes were blocked in a PBS buffer containing 0.1% Tween 20 supplemented with 5% skim milk (Sigma) or 3% BSA in the case of Strep-tag detection. For immunodetection, membranes were probed with polyclonal anti-CAT (abcam, 1:2,000), monoclonal anti-FLAG M2 (Sigma, 1:5,000), Strep-Tactin conjugated to horseradish peroxidase (IBA lifesciences, 1:10,000), DsRed polyclonal antibody (Takara, 1:2,000), anti-polyclonal anti-ssrAmk tag (1:2,000) or polyclonal antibodies specific to mycoplasma proteins (kind gift of Dr. Herrmann, Heidelberg University). These include anti-Lon (1:3,000) and anti-FtsH (1:3,000), which were also used as loading controls together with anti-CAT (abcam, 1:2,000) antibody. Anti-mouse IgG (1:10,000) or anti-rabbit IgG (1:5000) conjugated to horseradish peroxidase (Sigma) were used as a secondary antibody. Blots were developed with the Supersignal™ West Femto or West Pico Chemiluminescent Substrate detection Kit (Thermo Scientific) and signals detected in a LAS-3000 Imaging System (Fujifilm).
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3

Northern Blot Analysis of RNA

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Total RNA samples were electrophoresed at 4°C in 12% polyacrylamide 19:1 gels for siRNA and dsRNA ladder or in 1% agarose gels containing either 0.5X TBE buffer (44.5 mM Tris HCl, 44.5 mM boric acid, 1 mM EDTA, pH 8) or 1X HEPES buffer (20 mM HEPES, 1 mM EDTA, pH 7.4). Capillary transfer of nucleic acids was performed overnight onto a Hybond-N+ nylon membrane (GE Healthcare) with SSC 20X (3 M NaCl, 300 mM trisodium citrate adjusted to pH 7.0 with HCl) before UV-crosslink (Crosslinker UVC5000, Hoefer). Membranes were incubated in 5% skimmed milk powder for 2 h at room temperature to block nonspecific binding sites, incubated with a 3.5 μg.ml−1 B2 final concentration in PBST-milk for 1 h at room temperature, washed in PBST for 10 min three times and then incubated with a 1:5,000 dilution of Strep-Tactin conjugated to horseradish peroxidase from (IBA Lifesciences) in PBST-milk for 1h at room temperature. After washing in PBST for 10 min three times, membranes were incubated with Lumi-Light western blotting substrate (Roche Diagnostics) and revealed with Fusion FX imaging system (Vilber Lourmat) or Fujifilm autoradiographic films.
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