Assays were performed according to Patrone et al.19 (link) by q-PCR monitoring the incorporation of biotin-labeled triphosphate (biotin-16-UTP, 11388908910, Roche Applied Science) into RNA isolated on Streptavidin Magnetic Particles (11641778001, Roche Applied Science).
Streptavidin magnetic particles
Manufactured by Roche
Sourced in United States
Streptavidin magnetic particles are a type of lab equipment used in various biomedical applications. They consist of streptavidin, a protein with a high affinity for biotin, immobilized on the surface of magnetic beads. These particles can be used to capture and separate target molecules or particles that have been labeled with biotin.
Automatically generated - may contain errors
Lab products found in correlation
Chip seq library prep kits, by New England Biolabs (2 mentions)
Biotin 16 utp, by Roche (1 mentions)
Micro bio spin p 6 column, by Bio-Rad (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Ab7028, by Abcam (1 mentions)
Ab4729 100, by Abcam (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Proxeon easy nlc 1000 liquid, by Thermo Fisher Scientific (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ez link tfpa peg3 biotin kit, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
7 protocols using streptavidin magnetic particles
1
Biotin-labeled RNA Quantification
2
Biotinylated CMV DNA Template Assay
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Biotin labeled PCR products were generated using CMV_f and biotin labeled CMV_r. The biotin was linked to the 5′-end oligonucleotide via a 15-atom spacer (TEG, Operon) as described18 (link). 200 ng biotinylated CMV-DNA templates were bound to 10 μl of streptavidin magnetic particles (Roche) for 30 min and washed according to the manufacturer’s protocol. Immobilized CMV-DNA templates bound to the beads were then used for RNAP II transcription and pri-miRNA processing as performed with free CMV DNA templates. Bound and supernatant fractions were separated using a magnetic separator. Bound fractions were washed with 1 ml wash buffer (10 mM HEPES pH 7.6, 10% glycerol, 0.1 mM EDTA and 50 mM KCl) three times before RNA extraction.
3
Strand Exchange and FRET Assays
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All DNA sequences are shown in Supplementary Table 1 . For the strand exchange assay and EM, we used 80-mer ssDNA Oligo 1 for presynaptic filament assembly. To prepare the homologous duplex 40-mer dsDNA, Oligo 2 was 5’ end-labeled with [γ-32P] ATP (PerkinElmer) and treated with T4 polynucleotide kinase (New England Biolabs). Upon removal of the unincorporated nucleotide via a Micro Bio-Spin P-6 column (Bio-Rad), the radiolabeled oligonucleotide was then annealed to its complementary sequence (Oligo 3) by heating the mixture of these two oligonucleotides at 85 °C for 3 min and then slowly cooling it from 65 to 25 °C. The resulting duplex DNA was then purified from a 10% polyacrylamide gel and concentrated in TE buffer (10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA).
To prepare ssDNA streptavidin-magnetic beads for the DNA pulldown assay, 5′-end biotin-conjugated 80-mer ssDNA Oligo 1 was used to bind streptavidin-magnetic particles (Roche) as described previously64 (link).
For smFRET experiments, the fluorescing DNA overhang substrates were generated by using the paired oligos described in Supplementary Table1 and gradient PCR annealing from 85 °C to 25 °C in T + 50 buffer (20 mM Tris pH 8.0, 50 mM NaCl). These hybrid DNA substrates were then anchored to a slide surface-coated with biotin-mPEG and streptavidin66 (link).
To prepare ssDNA streptavidin-magnetic beads for the DNA pulldown assay, 5′-end biotin-conjugated 80-mer ssDNA Oligo 1 was used to bind streptavidin-magnetic particles (Roche) as described previously64 (link).
For smFRET experiments, the fluorescing DNA overhang substrates were generated by using the paired oligos described in Supplementary Table
4
ChIP-seq Profiling of Arid3a and Hdac1
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ChIP assays were performed with ES cell lines expressing BirA only (reference) or both BirA and biotin-tagged proteins (samples) as previously described (24 (link)) using streptavidin magnetic particles (Roche). Additional ChIP assays were carried out in ES, OEArid3a_ES+, OEArid3a_TS+ and TS cells using Arid3a (20 (link)), Hdac1 (ab7028, Abcam) and H3K27ac (ab4729–100, Abcam) antibodies. ChIP-seq libraries were generated using ChIP-seq library prep kits (NEB) according to the manufacturer's instructions. ChIP-seq libraries were sequenced using Illumina NextSeq 500 at the Genomic Sequencing and Analysis Facility (GSAF), The University of Texas at Austin.
5
Affinity Purification and Mass Spectrometry of Biotinylated Peptides
6
APEX2-Mediated Biotinylated Protein Enrichment
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Following the APEX2 labeling reaction, SK-N-SH cells were washed twice with a quenching solution and resuspended in 200 μL of ice-cold RIPA lysis buffer containing 25 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.16% sodium deoxycholate, 0.16% SDS, and supplemented with 1.5 mM EDTA, 5 mM Trolox, 10 mM L-ascorbate and protease inhibitors (cOmplete, Roche, Basel, Switzerland). After incubation for 30 min in a rotor at 4 °C, cell suspensions were centrifuged for 15 min at 14,000× g at 4 °C, and the supernatants (whole-cell lysates) were transferred into new tubes. Total protein amounts were then quantified by the Bradford Assay (BioRad Laboratories, Richmond, CA, USA).
Streptavidin magnetic particles (#11641786001, Roche) were equilibrated with RIPA lysis buffer by two washes. Each lysate was incubated with 90 μL of bead slurry in microcentrifuge tubes with rotation for 2 h at RT. The beads were subsequently washed twice with 1 mL RIPA lysis buffer, once with 1 mL of 1 M KCl, once with 1 mL of 100 mM sodium carbonate, twice with 1 mL of 2 M urea, and twice with 1 mL of RIPA lysis buffer. Biotinylated proteins were then eluted by incubating the bead slurry with 100 μL of 2 M thiourea, 6 M urea, 1% SDS, and 3 mM biotin in ddH2O for 15 min at RT followed by another 15 min incubation at 98 °C.
Streptavidin magnetic particles (#11641786001, Roche) were equilibrated with RIPA lysis buffer by two washes. Each lysate was incubated with 90 μL of bead slurry in microcentrifuge tubes with rotation for 2 h at RT. The beads were subsequently washed twice with 1 mL RIPA lysis buffer, once with 1 mL of 1 M KCl, once with 1 mL of 100 mM sodium carbonate, twice with 1 mL of 2 M urea, and twice with 1 mL of RIPA lysis buffer. Biotinylated proteins were then eluted by incubating the bead slurry with 100 μL of 2 M thiourea, 6 M urea, 1% SDS, and 3 mM biotin in ddH2O for 15 min at RT followed by another 15 min incubation at 98 °C.
7
ChIP-seq Profiling of Biotin-Tagged Proteins
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ChIP assays were done with ES cell lines expressing BirA only (reference) or both BirA and biotin-tagged proteins (samples) as previously described (Kim et al. 2008 (link)) using streptavidin magnetic particles (Roche). ChIP-seq libraries were generated using ChIP-seq library prep kits (New England Biolabs) according to the manufacturer’s instructions. ChIP-seq libraries were sequenced using an Illumina HiSeq 2500 at the Genomic Sequencing and Analysis Facility (GSAF) of The University of Texas at Austin. Raw and processed microarrays and ChIP-seq data have been deposited at the public server Gene Expression Omnibus (GEO) under accession number GSE56877.
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