HEK293T (cell line authenticated and tested without mycoplasma contamination) nuclear lysate is extracted using NE-PER™ Nuclear extraction kit from Thermo Scientific™. Streptavidin C1 magnetic beads were blocked by 4 ng/ml yeast tRNA and 50 μg/ml BSA buffer for 30 minutes in room temperature and nuclear lysate were pre-cleared by incubating with 20 μl prewashed Streptavidin C1 magnetic beads (Thermo Scientific™) for 1 h in 4°C. Three hundred pmol oligo and 60 μl prewashed Streptavidin C1 magnetic beads were added for each reaction and the 5′ Biotin-labeled rG4 wildtype or mutant oligos were heated at 75°C for 5 min for denaturation before adding to the mixture containing buffer A (10 mM Tris–HCl pH 7.5, 100 mM KCl, 0.1 mM EDTA, with/without 20 μM PDS). The beads immobilization step is processed in room temperature for 30 minutes in buffer A. The beads were washed for 3 times with buffer A. And 500 μg nuclear lysate was added in each reaction to the incubation mixture containing buffer B (20 mM Tris–HCl pH 7.5, 50 mM KCl, 0.5 mM EDTA, 10% glycerol). The incubation step is processed in 4°C for 2 h and followed by 5 times stringent washes using buffer B. The enriched protein were eluted by heating at 95°C for 30 min in 1× Laemmli sample buffer (BioRad) and processed to western blot. The data was quantified by ImageJ and analyzed with Microsoft Excel.
Streptavidin c1 magnetic beads
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Streptavidin C1 magnetic beads are a type of laboratory equipment used in various biomedical applications. They consist of superparamagnetic particles coated with the protein streptavidin, which has a high affinity for the molecule biotin. These beads can be used to isolate and purify biomolecules, such as proteins or nucleic acids, that are tagged with biotin.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
Co ip buffer, by Beyotime (2 mentions)
Rnase free dnase 1, by Takara Bio (2 mentions)
T7 rna polymerase, by Roche (2 mentions)
T4 rna ligase 1, by Qiagen (2 mentions)
Biotin rna labeling mix, by Roche (2 mentions)
Superase in, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Hiseq 4000 platform, by Illumina (2 mentions)
Kapa hyper pre kit, by Roche (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Truseq chip sample preparation kit, by Illumina (2 mentions)
Proteinase k, by Merck Group (2 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Ne per nuclear extraction kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Non reducing lane marker sample buffer, by Thermo Fisher Scientific (1 mentions)
Protector rnase inhibitor, by Roche (1 mentions)
Pierce phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Tripletof 6600 system, by AB Sciex (1 mentions)
60 protocols using streptavidin c1 magnetic beads
1
Affinity Purification of G4-Binding Proteins
2
Probing Circular RNA-Protein Interactions
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Cultured cells (107) were lysed in 500 μl co-IP Buffer (Cell lysis buffer for western and IP, Beyotime, China), supplemented with protease inhibitor cocktail Tablets (Roche, Switzerland), PierceTM phosphatase inhibitor (Thermo Scientific), and Protector RNase Inhibitor (Roche). Next, the cell lysates were incubated with 800 pmol of biotinylated DNA probes for circ_CEA (5′-GCCCATCAGTCTTCCTGAAA-3′) or scramble probes (5′-ATCTAATAGCTCCACGTGCC-3′) at 4 °C overnight. Next, Streptavidin C1 magnetic beads (Invitrogen) were blocked with 2 mg/mL BSA at room temperature for 1 hr, and then added to each binding reaction and incubated at room temperature for 1 hr. After washing in co-IP Buffer, beads were incubated with Non-Reducing Lane Marker Sample Buffer (Thermo Scientific) at room temperature for 10 min to elute the bound proteins. The proteins were detected by western blotting with the antibodies against p-p53 ser315, p53, CDK1, and FoxO3 (Cell Signaling Technology).
3
Biotin-labeled RNA Pulldown for circPTK2
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Biotin-labeled RNA for liner sequence of circPTK2 was generated by an in vitro transcription reaction with the Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche, Mannheim, Germany), and then treated with RNase-free DNase I (Takara, Japan). After incubation with guide oligonucleotide targeting circular junction, the liner probe was then circularized using T4 RNA ligase I, treated with RNase R. After purified with RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA), the biotin-labeled RNA probe (3 μg) was then incubated with cell extracts from CRC cells at room temperature (RT) for 2 h, and treated with 35 μl of Streptavidin C1 magnetic beads (Invitrogen) for 1 h. after washed, the retrieved protein was detected by western blot or mass spectrometry analysis (CapitalBio Technology, Beijing, China).
4
Biotinylated circNR3C1 Probe Enrichment
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The biotinylated probe of circNR3C1 was synthesized by TSINGKE (Beijing, China). The sequence of the probe was listed in Table S1 . In brief, lysates of 1 × 107 BC cells were incubated with biotin-labeled linear or circular probe for 4 h and treated with appropriate Streptavidin C1 magnetic beads (Invitrogen) for 30 min. After washed thoroughly with wash buffer for three times, the retrieved protein was detected by western blot or MS analysis at Wuhan Institute of Biotechnology (Wuhan, China).
5
RNA Pull-Down Assay for miR-552-3p
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RNA pull‐down assays were conducted as described previously30 , 31 . In short, 1 × 107 cells were washed in ice‐cold phosphate‐buffered saline, lysed in 500 μL co‐IP buffer, and then incubated with 3 μg of biotin‐labeled miR‐552‐3p probe to bind endogenous miR‐552‐3p at room temperature for 2 h. Next, 50 μL of washed Streptavidin C1 magnetic beads (Invitrogen) was added to each binding reaction and incubated at room temperature for 50 min; after which, the beads were carefully washed with co‐IP buffer. Finally, the RNA‐binding protein LXRa was detected by western blotting.
6
Biotinylated DNA Probe Pulldown Assay
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The pull-down assay was performed as described in previous study.9 (link) In brief, 107 cells were washed with cold PBS, lysed in 500 μL co-IP buffer, and incubated with 3 μg biotinylated DNA oligo probes against endogenous or ectopically expressed circ-Foxo3, at room temperature for 2 h. A total of 50 μL washed Streptavidin C1 magnetic beads (Invitrogen) were added to each binding reaction for 1 h at room temperature. Then, the RNAs were purified using RNeasy Mini Kit (QIAGEN) for further qPCR analysis.
7
Identification of circAnks1a-binding Proteins
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The full-length sequence of circAnks1a was constructed into a linear DNA template containing the T7 promoter. A biotinylated RNA probe was transcribed in vitro using T7 RNA polymerase (Thermo Scientific) and bound to streptavidin C1 magnetic beads (Invitrogen). Spinal dorsal horn tissues were ground in liquid nitrogen and incubated in lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2, 1% NP40, SUPERase-In (Ambion), and protease inhibitors (Roche)] on ice for 30 min. The lysates were then incubated with the RNA probe for 2 h at 4 °C. The beads were briefly washed five times with wash buffer and boiled in sodium dodecyl sulphate (SDS) buffer. The retrieved proteins were separated via SDS-PAGE. The gel bands were manually excised and digested with mass spectrometry-grade trypsin (Promega). The digested peptides were analyzed on an AB Sciex TripleTOF® 6600 System (AB Sciex). The mass spectrometry data were analyzed and identified using Mascot (Matrix Science) in the NCBI Rattus database and the UniProt Rattus database.
8
Biotin-Labeled RNA Probe Pulldown Assay
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The biotin-labeled RNA probe and control probe were in accordance with those used in the FISH assay. Modified cells (1 × 107) were transfected with biotin-labeled probes for 24 h and then lysed in IP buffer containing RNase inhibitor. The lysates were incubated with 50 µL of Streptavidin C1 magnetic beads (Invitrogen, USA) at room temperature for 30 min and then washed briefly with IP buffer, followed by western blotting and qRT-PCR.
9
RNA Pull-Down Assay for circNDUFB2
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For RNA pull-down assay, 1 × 107 cells were washed in ice-cold PBS, lysed in 500 μl co-IP buffer (Thermo Scientific) supplemented with a cocktail of proteinase inhibitors, phosphatase inhibitors, and RNase inhibitor (Invitrogen), and then incubated with 3 μg biotinylated DNA oligo probes against circNDUFB2 backsplice junction region (sense) or corresponding complementary probes (antisense) for 2 h at room temperature. A total of 50 μl washed Streptavidin C1 magnetic beads (Invitrogen) were added to each binding reaction and further incubated for another hour at room temperature. The beads were washed briefly with co-IP buffer for five times. Finally, the retrieved proteins were used for mass spectrometry or western blot analysis. Probe sequences are listed in Supplementary Data 4 .
10
Biotin Probe Identification of Circular RNA Interactome
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A biotin probe targeting the back-splicing site of circ_0006156 was designed by GenePharma (China). The probe was incubated with extracts from the indicated cells at room temperature for 2 h, and Streptavidin C1 magnetic beads (35 μL, Invitrogen) were then added for 1 h. After washing, the protein was detected by Western blot and mass spectrometry analyses (Bioprofile, China). The sequences of probes can be seen in Supplemental Table 2 .
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