SELEX was performed as previously described34 (link). Briefly, a T7 promoter-driven library of 92,918 scrambled 110mer oligos was synthesized by CustomArray Inc®. The DNA oligo library was PCR amplified, followed by in vitro transcription using the MEGAscript T7 Transcription Kit (Thermo Fisher®) as per the manufacturer’s instructions. RNA was heated at 65°C for 5 min and immediately put on ice before the selection process. Flag-tagged proteins were purified from RNaseIII−/− cells at 24 hpt in lysis buffer (15 mM Tris/HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2, and 1% (v/v) TritonX-100) supplemented with 1x protease inhibitor cocktail (Roche®). Cell lysates were incubated with anti-Flag affinity resin (Sigma A2220) at 4 °C overnight, followed by four washes 5 min each with lysis buffer. 1 μg of in vitro transcribed RNA in lysis buffer was added to the beads and incubated at 4 °C for 2 hrs, followed by four washes 5 min each with lysis buffer. Protein-RNA complexes were eluted with 300 μg/ml Flag-peptide (Sigma F3290) and purified RNA was extracted using Trizol. Purified RNA was reverse transcribed, PCR amplified and in vitro transcribed to repeat the selection process. A total of 5 rounds of selection were performed.
Anti flag affinity resin
Manufactured by Merck Group
The Anti-Flag affinity resin is a chromatography medium designed for the purification of recombinant proteins tagged with the Flag epitope. The resin is composed of agarose beads coupled with monoclonal antibodies specific to the Flag tag, allowing for the selective capture and isolation of Flag-tagged proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by Merck Group (7 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Ab94010, by Abcam (2 mentions)
Maxiscript kit, by Thermo Fisher Scientific (2 mentions)
Tnt7 transcription translation kit, by Promega (2 mentions)
Ribonucleoside vanadyl complex, by New England Biolabs (2 mentions)
Protease inhibitor cocktail, by New England Biolabs (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Anti ha affinity resin, by Merck Group (1 mentions)
Type 45 ti rotor, by Beckman Coulter (1 mentions)
Superose 6 increase 3.2 300, by GE Healthcare (1 mentions)
6875 freezer mill, by SPEX (1 mentions)
Anti ha agarose, by Merck Group (1 mentions)
3xflag peptide, by Merck Group (1 mentions)
Rabbit reticulocyte lysate, by Promega (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Transit 293, by Mirus Bio (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Hydroxyproline, by Abcam (1 mentions)
17 protocols using anti flag affinity resin
1
SELEX Protocol for Protein-RNA Interactions
2
Affinity Purification of Protein Complexes
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The Sf21 cells expressing recombinant double HA-tagged Elg1 (2xHA-Elg1), His-Flag-Br140, His-Flag-Enok and/or no-tag Br140 were lysed in lysis buffer (50 mM HEPES at pH 7.5, 2 mM MgCl2, 300 mM NaCl, 10% glycerol, 0.5 mM EDTA, 0.2% Triton X-100, 1 mM PMSF, 1 µg/mL pepstatin A, 1 µg/mL leupeptin, 1 µg/mL aprotinin) for 20 min on ice. After centrifugation at 20,000g for 10 min at 4°C, supernatants were collected and incubated with anti-Flag affinity resin or anti-HA affinity resin (Sigma, E6779) for 3 h at 4°C. Following three washes with lysis buffer, resins were heated in 1× SDS sample buffer for 6 min at 98°C and subjected to Western blot analysis.
3
Electrophoretic Mobility Shift Assay
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EMSAs were performed as previously described35 (link). Flag-tagged proteins were purified from RNaseIII−/− cells at 24 hpt in lysis buffer (15 mM Tris/HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2, and 1% (v/v) TritonX-100) supplemented with 1x protease inhibitor cocktail (Roche®). Cell lysates were incubated with anti-Flag affinity resin (Sigma A2220) at 4 °C overnight, followed by four washes at 5 min each with lysis buffer. Bound protein was eluted with 300 μg/ml Flag-peptide (Sigma F3290) in 20 mM Tris/HCl pH 8.0, 100 mM KCl, and 0.2 mM EDTA. Alternatively, a fragment of recombinant human Drosha protein was purchased from Abcam (ab94010) and 0.25–1 μg of this purified protein was used for EMSAs. Radioactively labeled RNA was in vitro transcribed from a PCR-amplified template using the MAXIscript kit (Ambion®). For complex formation of protein and RNA, protein eluate or recombinant protein was incubated with radio-labeled RNA (~100,000 cpm per reaction) for 30 min at room temperature in 20 mM Tris/HCl pH 8.0, 100 mM KCl, and 0.2 mM EDTA, supplemented with 0.1 μg/μl BSA and 1 mM DTT. Reactions were run on a 6% polyacrylamide gel.
4
SELEX Protocol for Protein-RNA Interactions
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SELEX was performed as previously described34 (link). Briefly, a T7 promoter-driven library of 92,918 scrambled 110mer oligos was synthesized by CustomArray Inc®. The DNA oligo library was PCR amplified, followed by in vitro transcription using the MEGAscript T7 Transcription Kit (Thermo Fisher®) as per the manufacturer’s instructions. RNA was heated at 65°C for 5 min and immediately put on ice before the selection process. Flag-tagged proteins were purified from RNaseIII−/− cells at 24 hpt in lysis buffer (15 mM Tris/HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2, and 1% (v/v) TritonX-100) supplemented with 1x protease inhibitor cocktail (Roche®). Cell lysates were incubated with anti-Flag affinity resin (Sigma A2220) at 4 °C overnight, followed by four washes 5 min each with lysis buffer. 1 μg of in vitro transcribed RNA in lysis buffer was added to the beads and incubated at 4 °C for 2 hrs, followed by four washes 5 min each with lysis buffer. Protein-RNA complexes were eluted with 300 μg/ml Flag-peptide (Sigma F3290) and purified RNA was extracted using Trizol. Purified RNA was reverse transcribed, PCR amplified and in vitro transcribed to repeat the selection process. A total of 5 rounds of selection were performed.
5
Purification of Trs120-3xFLAG Protein
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The Trs120-3xFLAG–tagged S. cerevisiae (44 liters) were grown in YPD (yeast extract peptone dextrose) medium at 30°C overnight until an OD600 (optical density at 600 nm) reached 5 to 6. The cells were pelleted by centrifugation and resuspended using the lysis buffer [20 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 10% glycerol, 1% CHAPS, 1 mM EDTA, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF)] supplemented with a protease inhibitor cocktail (Roche). The cell pellets were frozen by liquid nitrogen and then lysed with Freezer Mill 6875 (SPEX CertiPrep). The lysate was centrifuged at 142,400g for 40 min using a Type 45 Ti rotor (Beckman). All supernatants were incubated with anti-Flag affinity resin (Sigma-Aldrich) at 4°C for 2 hours. The resin was then washed by the washing buffer [20 mM Hepes-NaOH (pH 7.4), 300 mM NaCl, 10% glycerol, 0.1% CHAPS, 1 mM EDTA, 2 mM DTT] and eluted with the washing buffer supplemented with Flag peptide (0.1 mg/ml; Sigma-Aldrich). The eluate was concentrated and applied to Superose 6 Increase 3.2/300 (GE Healthcare) equilibrated with the sample buffer [20 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 2 mM DTT, 0.05% digitonin]. The peak fractions were analyzed by SDS-PAGE and the MS analysis.
6
In Vitro Synthesis of HA-tagged NS1 Variants
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HA-tagged NS1 variants were synthesized in vitro using the pcDNA3 plasmids and the TNT7 transcription/translation kit (Promega) by following the manufacturer's recommendations. Human 293T cells (6-well format; 1.5 ×106 cells/well) were transiently transfected with 8,000 ng of a pCAGGS plasmid expressing a FLAG-tagged version of the human CPSF30 (17 (link)). At 30 hpt, cells were lysed in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 5% glycerol, and 1% Triton X-100 and supplemented with a cOmplete mini protease inhibitor cocktail (Pierce). Cleared cell lysates expressing FLAG-CPSF30 were incubated overnight at 4°C with the in vitro-synthesized NS1 proteins and 20 μl of an anti-FLAG affinity resin (Sigma-Aldrich). After extensive washing, precipitated proteins were dissociated from the resin using disruption buffer and analyzed by Western blotting as described above using rabbit anti-HA (NS1)- and anti-FLAG (CPSF30)-specific polyclonal antibodies.
7
Recombinant Protein Expression and Purification
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APP‐C99 in pcDNA5, PPL‐C99 in pcDNA3.1 and SGTA in pHisTrx were previously described 17 whilst PPLssKO‐C99 and PPL‐C99 lysine mutants in pcDNA3.1, SGTAD27R/E30R and SGTAK160E/R164E in pHisTrx were prepared by site‐directed mutagenesis. Plasmids coding for proteins used in Fig EV3 were obtained from SinoBiological. Recombinant proteins were purified as before 17 , 39 , 66 . Mouse anti‐SGTA (clone 47‐B), anti‐SRP54 (clone 30) and chicken control IgY antibodies were purchased from SantaCruz Biotechnology whilst mouse anti‐β‐amyloid (clone BAM‐10), mouse control antibodies, anti‐FLAG affinity resin, anti‐HA agarose and 3xFLAG peptide from Sigma. Rabbit anti‐Hbs1L antibody (A305‐395A) was from Bethyl Laboratories whilst chicken anti‐SGTA, rabbit anti‐C99 and rabbit anti‐Bag6 antibodies were made to order 17 . Rabbit reticulocyte lysate (RRL) for in vitro protein translation was from Promega, chemical cross‐linkers from Thermo Fisher Scientific, and ε‐TDBA‐Lys‐tRNA was made essentially as previously described 48 .
8
Immunoprecipitation and Protein Isolation
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The proteins were immunoprecipitated by incubating the lysate (1 mg) with 20 μl anti-FLAG affinity resin (Sigma-Aldrich) for 2 h at 4 °C. The resin was washed with 25 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% nonionic polyoxyethylene-40, 100 μM phenylmethylsulfonyl fluoride, 100 μM sodium orthovanadate, 5 μg/ml aprotinin, and 5 μg/ml leupeptin.
9
Transient Co-transfection of IFI27 and RIG-I
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Human 293T cells (100 mm-plate format) were transiently co-transfected with the plasmids pCAGGS-IFI27-HA alone or together with pCAGGS-RIG-I-FLAG, using lipofectamine 3000 (ThermoFisher Scientific), for 24 h. The total amount of transfected DNA plasmid was maintained constant by co-transfecting the empty pCAGGS plasmid when needed. Later, cells were transfected with poly(I:C) (1,000 ng/ml) using PEI for an additional 24 h or were infected with SeV (MOI 3) during 24 h, and cells were lysed in the Co-IP buffer and cleared by centrifugation. Where indicated, cellular lysates were treated with RNaseA (10 U/ml), RNase T1 (400 U/ml) and RNAse III (10 U/ml), during 30 min at 37°C, as previously reported (Laraki et al., 2008 (link)). Cleared cell lysates were incubated overnight at 4°C with the anti-FLAG affinity resin (Sigma-Aldrich, A2220). Then, the mixtures were washed three times in TBS buffer containing 0.1% SDS, and precipitated proteins were dissociated using 0.1 M glycine buffer at pH 2.4, denatured in loading buffer and incubated at 95°C, during 5 min. Then, samples were analyzed by electrophoresis and Western blot as described above using anti-HA (IFI27), and anti-FLAG (RIG-I) specific Abs.
10
Protein-Protein Interaction Assay
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In vitro co-IP was performed for purified C1QTNF4 and purified nucleolin constructs to assess direct interaction. For IP with FLAG affinity resin, 50 nM R1234G was mixed with 200 nM C1QTNF4 and incubated overnight at 4 °C on an end-to-end rotator. For IP by GFP-Trap Agarose, 50 nM C1QTNF4 constructs were mixed with 200 nM nucleolin constructs in 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 5% glycerol and incubated for 2 h at 4 °C on an end-to-end rotator. In total, 10 μl washed anti-FLAG affinity resin (Sigma-Aldrich) or 5 μl washed GFP-Trap Agarose was added and incubated for a further 2 h at 4 °C. The beads were collected and washed three times in 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 5% glycerol. Bound protein was eluted by boiling in reducing 2X Laemmli sample buffer at 95 °C for 10 min. Samples were analyzed by immunoblot.
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