RNA pull-down analysis was performed as previous report 15 (link). In brief, Full-length AY or mutated AY RNA was in vitro transcribed using T7 RNA polymerase and labeled with biotin (Roche, Mannhein, Germany). Then cytoplasmic extracts prepared from Hep3B cells using RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 0.2% SDS, 1% NP40, 1% Triton X-100, 1mM EDTA, and 50 mM Tris pH 8.0) were incubated with in vitro transcribed and biotinylated RNA, which were then targeted with streptavidin beads (Vector Laboratories, CA, USA) and washed. The associated proteins were resolved by gel electrophoresis. RNA immunoprecipitation assays were performed as reported previously 9 (link). Hep3B cells were treated with 1% formaldehyde, dissolved in RIPA buffer, and supplemented with Recombinant RNase Inhibitor (Takara, Dalian, China) and Protease Inhibitor Cocktail (Millipore, MA, USA). The lysates were sonicated and then centrifuged at 13,800 g for 10 min. After preclearing, supernatants were incubated with indicated antibodies for 4 hours and subsequently incubated with protein G agarose (Millipore, MA, USA) for 2 hours. AY enrichment was analyzed using qRT-PCR. Antibody information used in these 2 assays is summarized in Supplemental Table 2 .
Streptavidin beads
Manufactured by Vector Laboratories
Sourced in United States
Streptavidin beads are solid-phase affinity matrices composed of streptavidin covalently coupled to agarose or magnetic beads. Streptavidin has a high affinity for biotin, allowing these beads to be used for the capture, isolation, and purification of biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Gsl 2 biotinylated, by Vector Laboratories (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Recombinant rnase inhibitor, by Takara Bio (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (1 mentions)
Protein a g sepharose beads, by Thermo Fisher Scientific (1 mentions)
Tripletof 5600, by AB Sciex (1 mentions)
6 protocols using streptavidin beads
1
RNA Pull-Down and Immunoprecipitation Analysis
2
Immunoprecipitation for Protein Identification
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For the immunoprecipitation (IP) assay, cell lysates (0.8 mg) were incubated with 4 µg of antibody at 4 °C for 16 h. Protein L sepharose beads (BioVision Inc., Milpitas, CA, USA) were then added to the lysates for 3 h for pulldown of the CS56 antibody (mouse IgM). For protein identification, pull-downed proteins were digested by trypsin, and applied to tandem mass spectrometry (LC MS/MS, Dionex Ultimate 3000 RSLCnano system hybrid mass spectrometer). The data files obtained following LC-MS/MS analysis were processed in SwissProt, Mascot version 2.5, and Percolator1,2.
Protein A/G sepharose beads (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to capture CD44 antibody (mouse IgG). The CD44 IP proteins were then separated by 8% SDS-PAGE, and applied to Western blotting. For the C6S-p pulldown assay, a total 0.8 mg of cell lysate was mixed with C6S-p gently and incubated at 4 °C for 16 h. Streptavidin beads (Vector Laboratories, SA-5010, Burlingame, CA, USA) were added to the lysate and incubated for 3 h. The bead pulldown samples were analyzed by Western blotting.
Protein A/G sepharose beads (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to capture CD44 antibody (mouse IgG). The CD44 IP proteins were then separated by 8% SDS-PAGE, and applied to Western blotting. For the C6S-p pulldown assay, a total 0.8 mg of cell lysate was mixed with C6S-p gently and incubated at 4 °C for 16 h. Streptavidin beads (Vector Laboratories, SA-5010, Burlingame, CA, USA) were added to the lysate and incubated for 3 h. The bead pulldown samples were analyzed by Western blotting.
3
Biotinylated RNA Pulldown Proteomics
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RNA pull‐down analysis was performed as previously described.27 In brief, BC was transcribed in vitro using T7 RNA polymerase, labelled with biotin (Roche, Mannheim, Germany) and then incubated with cell protein extracts, which were then conjugated with streptavidin beads (Vector Laboratories, CA, USA) and washed. The bead‐associated proteins were resolved by SDS‐PAGE, and then the stained protein bands were cut from the gel and transferred into 1.5 ml EP tubes. After centrifugation and drying for 15 min, 10 μl trypsin (20 ng/μl, dissolved in 25 mmol/L NH4HCO3) was added and digested at 37°C overnight. The lysate were dissolved in 2%ACN/98%H2O/.1%FA solution and analysed by LC‐LTQ‐MS (TripleTOF 5600, AB Sciex, Framingham, MA). In addition, the spray voltage was 2.2 kV, whereas the temperature of capillary electrophoresis was 200°C. Data were analysed using ProteinPilot software V4.4 (AB Sciex, Framingham, MA).
4
Lectin Pull-Down Assay Protocol
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For the lectin pull-down assay, cell lysates (1 mg) were incubated with Griffonia Simplicifolia Lectin II (GSL-II) biotinylated (Vector Laboratories) at 4 °C for 16 h. The next day, the sample was incubated with streptavidin beads (Vector Laboratories) for 2 h at room temperature. Finally, the pull-down proteins were analyzed via western blot analysis.
5
Lectin Pull-Down Assay Protocol
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For the lectin pull-down assay, cell lysates (1 mg) were incubated with Griffonia Simplicifolia Lectin II (GSL-II) biotinylated (Vector Laboratories) at 4 °C for 16 h. The next day, the sample was incubated with streptavidin beads (Vector Laboratories) for 2 h at room temperature. Finally, the pull-down proteins were analyzed via western blot analysis.
6
Quantifying miRNA Expression by RNA Pull-Down
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RNA pull-down analysis was performed as previous report [11 (link)]. Then cytoplasmic extracts prepared from MG-63 and 143B cells using RIPA buffer were incubated with in vitro transcribed and biotinylated RNA, which were then targeted with streptavidin beads (Vector Laboratories, CA, USA) and washed. After purifified, Real-time PCR was performed to examine the expression levels of selected miRNAs.
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