Protein identification was performed with an adapted protocol described previously by Berezovski et al.41 (link) To isolate protein targets, 6 million GSCs (#1 and #83) were lysed with sodium deoxycholate (Sigma-Aldrich) at 0.1% (w/v) in 10 mM PBS with Ca2+, Mg2+ (Sigma-Aldrich) and incubated with a biotinylated-scrambled oligonucleotide (TriLinK Biotechnologies, San Diego, CA, USA) at 200 nM for 30 min at room temperature, as a counterselection step. Then, lysates were incubated with streptavidin MagneSphere paramagnetic particles (Promega, Milan, Italy) for 30 min at 4°C. Afterward, unbound proteins were incubated with biotinylated A40s (TriLinK Biotechnologies) at 200 nM for 30 min at room temperature. A40s-protein complexes were incubated with streptavidin MagneSphere paramagnetic particles (Promega) for 30 min at 4°C. Collected beads were washed twice with cold 10 mM PBS with Ca2+, Mg2+ (Sigma-Aldrich). Thus, incubation with 8 M urea for 1 h at 4°C led to protein denaturation and release from the aptamer and magnetic beads. Pull-down proteins were separated by SDS-PAGE (10% polyacrylamide gel), transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and immunoblotted for EphA2 antibody (Santa Cruz Biotechnology).
Streptavidin magnesphere paramagnetic particles
Manufactured by Promega
Sourced in United States
Streptavidin MagneSphere Paramagnetic Particles are magnetic beads coated with the streptavidin protein. Streptavidin has a high affinity for biotin, allowing the particles to be used for the separation and purification of biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Rnasin, by Promega (2 mentions)
Epha2 antibody, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Ca2 mg2, by Merck Group (1 mentions)
Pfastbachtb, by Thermo Fisher Scientific (1 mentions)
Fl 218, by Santa Cruz Biotechnology (1 mentions)
Ab32381, by Abcam (1 mentions)
Ecorv, by New England Biolabs (1 mentions)
Bglii, by New England Biolabs (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Alexa fluor 488 conjugated goat anti human igg, by Jackson ImmunoResearch (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 trihydrochloride, by Thermo Fisher Scientific (1 mentions)
Nta chip, by GE Healthcare (1 mentions)
Sypro orange, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Hitrap mabselect sure 5 ml column, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
30 protocols using streptavidin magnesphere paramagnetic particles
1
Protein Identification by Aptamer Pulldown
2
Biotinylated EV71 5'UTR RNA Binding Assay
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The reaction mixtures contained 300 μg cell extracts and 3 μg biotinylated EV71 5′UTR RNA probe. Final reaction mixture volumes were adjusted to 100 μl with RNA mobility shift buffer (5 mM HEPES, 40 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 2 mM dithiothreitol, 1 U RNasin and 0.25 mg/ml heparin). The mixtures were incubated for 15 min at 30°C, and 400 μl Streptavidin MagneSphere Paramagnetic Particles (Promega) were added for binding at room temperature for 10 min. The RNA–protein complexes were washed six times with RNA mobility shift buffer (5 mM HEPES, 40 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 2 mM dithiothreitol, 1 U RNasin). Then 32 µl 1×PBS and 8 µl 5×SDS-PAGE loading buffer were added to reaction mixtures. Samples were boiled for 5 min, subjected to 12% SDS-PAGE, and then transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in 5% skim milk in 1×PBST buffer, washed three times with 1×PBST buffer, and incubated with the anti-SIRT1 antibody overnight at 4°C. The membranes were washed three times with 1×PBST (PBS with 0.04% Tween 20) buffer, treated with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit antibody for 45 min at room temperature, and washed six times with 1×PBST buffer. The membranes were incubated with HRP substrate, and then we proceeded with HRP color development.
3
Transcription Arrest and Backtrack Cleavage
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Biotinylated template was bound to Promega Streptavidin MagneSphere Paramagnetic Particles. Transcription was initiated in the presence of Qλ and allowed to proceed for 2 min to form the Qλ-dependent arrested complexes (QAC), at which point the reactions were washed three times with T buffer + 0.1 mg/ml bovine serum albumin to remove nucleotides and magnesium chloride, halting the reaction. For nucleotide starvation cleavage, reactions were then resuspended in T buffer + 100 nM GreB, incubated at 37°C, and initiated by addition of magnesium chloride to 5 mM. To cleave backtracked RNAs and chase the 5′ cleavage products, reactions were resuspended in T buffer, 100 nM GreB and 200 μM NTPs, incubated at 37°C, and initiated by addition of magnesium chloride to 5 mM and rifampicin to 10 μg/ml. Reactions were stopped by adding 125 μl of stop solution.
4
EV71 5'-UTR RNA-Protein Binding Assay
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Reaction mixtures contained 200 µg of cell extract proteins and 3 µg of biotinylated EV71 5′-UTR RNA (or fragments thereof). The reaction mixture’s final volume was adjusted to 100 µl with RNA mobility shift buffer [5 mM HEPES (pH 7.1), 40 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 2 mM dithiothreitol, 1 U RNasin and 0.25 mg/ml heparin]. The mixtures were incubated for 15 min at 30°C and then added to 400 µl of Streptavidin MagneSphere Paramagnetic Particles (Promega) for binding at room temperature for 10 min. Beads, containing RNA-protein complexes, were washed five times with RNA mobility shift buffer lacking heparin. After the last wash, 15 µl of 6×SDS-PAGE sample buffer was added to the beads and the mixtures were incubated for 10 min at room temperature. The eluted proteins were boiled and fractionated by 10% SDS-PAGE. Specific proteins were detected by Western blot analyses as described below. Expression and purification of recombinant His6-p40AUF1 for competition assays was described previously [36] (link).
5
Monobody Screening of MLKL Pseudokinase
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For monobody screening, bait protein was generated from a bacmid prepared in DH10MultiBac E. coli (ATG Biosynthetics) from a pFastBac Htb (Thermo) vector encoding a TEV (tobacco etch virus) protease-cleavable His6 fusion N-terminal to the human MLKL pseudokinase domain (residues 190–471), which was C-terminally fused to a non-cleavable flexible penta-Ser linker and AviTag (ASSSSSGLNDIFEAQKIEWHE) using the Bac-to-Bac system in Sf21 insect cells, as reported for untagged counterparts25 (link),36 (link),45 (link). The AviTag of human MLKL pseudokinase domain was biotinylated using recombinant His-BirA using established protocols31 (link), and biotinylated protein eluted from a Superdex S200 10/300 GL size-exclusion chromatography column in 0.2 M NaCl, 20 mM HEPES pH 7.5, 5% v/v glycerol. The bait was immobilized by binding to Streptavidin MagneSphere Paramagnetic Particles (Promega) and subjected to four rounds of screening of monobody phage-displayed libraries, before enriched sequences were subcloned into a yeast display library and subjected to two further rounds of yeast displayed screening, using established procedures31 (link),39 (link),62 (link).
6
EV71 5'UTR RNA-Protein Interactions
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RD cell extracts (200 μg), 12.5 pM of a biotinylated EV71 5′ UTR RNA probe, and recombinant PTBP1, FUBP1 and PCBP2 were mixed together, and the mixture (with a final volume of 100 μl), which contained 5 mM HEPES pH 7.1, 40 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 2 mM DTT and 0.25 mg/ml heparin (RNA mobility shift buffer), was incubated for 15 min at 30°C, and then added to 400 μl of Streptavidin MagneSphere Paramagnetic Particles (Promega) for binding at room temperature for 10 min. The protein-RNA complexes were washed five times with heparin-free RNA mobility shift buffer, after which 30 μl of 2× sample buffer was added to the beads and allowed to incubate for 10 min at room temperature. The sample containing the eluted proteins was incubated at 95°C for 5 min and resolved on a 12% gel by SDS-PAGE. The interactions were detected by western blot using specific antibodies.
7
Affinity Purification of RNA-Binding Proteins
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HL-60 cells were treated with VitD for 72 hours and 30 × 106 cells were collected and washed with ice cold PBS and resuspended in Buffer A (20 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl, 0.1% NP40, 0.2 mM EDTA, 10% glycerol). 800 μg of supernatant (cytoplasmic extract) were incubated with tRNA-saturated magnetic beads (Streptavidin MagneSphere Paramagnetic Particles, Promega) and ten biotinylated DNA probes in IP-Buffer (20 mM Hepes KOH pH 7.4, 150 mM KCl, 2 mM MgCl2, 0.01% Triton X-100, 5% glycerol, 1 mM DTT) for 1 hrs at R.T. Beads were washed four times with IP-Buffer and resuspended in 1 mL of Trizol and Protein loading dye for RNA and protein analysis, respectively. Protein levels were analyzed by western blot as previously described (Hughes et al., 2015). Antibodies utilized were against Ago2 (ab32381, Abcam) and HPRT (FL-218, Santacruz Biotechnology).
8
Synthesis of DNA templates for RNAPII transcription
9
Comprehensive Molecular Biology Protocol
10
Affinity Purification of Non-Denaturing Proteins
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Non-denaturing proteins of PK cells and LM of the pig were extracted by Non-denaturing Lysis Buffer (Sangon). Later, we typically bonded non-denaturing proteins and biotin-labeled DNA probes by rotation, which were then supplemented with Streptavidin MagneSphere® Paramagnetic Particles (Promega). Then, the reactions were further rotated and washed. Later, DNA-bound proteins were collected with 10% SDS (sodium dodecyl sulfate, sodium salt) and analyzed by Western blotting, taking non-denaturing proteins/Streptavidin MagneSphere® Paramagnetic Particles as positive/negative control.
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