Biotin-labeled miR-127-5p probe and control probe were synthesized by Sangon Biotech (Shanghai, China). Probe-coated beads were generated by co-incubating the probe with streptavidin-coated beads (Invitrogen, CA, USA) at a temperature of 25°C for 2 hours. ESCC cells were collected and lysed and incubated with miR-127-5p probes overnight at 4°C. Following this, the beads were eluted and the complex was purified with TRIzol (Takara, Dalian, China). Then the abundance of circNELL2 and CDC6 was analyzed using qRT-PCR.
Streptavidin coated beads
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Streptavidin-coated beads are a type of laboratory equipment used in various biotechnology applications. Streptavidin, a protein derived from the bacteria Streptomyces, is covalently bound to the surface of the beads. The primary function of these beads is to enable the capture and separation of biotinylated molecules, such as proteins, nucleic acids, or cells, from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Takara Bio (6 mentions)
Control probe, by Sangon (3 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (3 mentions)
Sureselectxt human all exon v4, by Agilent Technologies (2 mentions)
Herculase 2, by Agilent Technologies (2 mentions)
Agilent sureselectxt humanallexon v4 50 mb, by Agilent Technologies (2 mentions)
Sureselect library preparation kit, by Agilent Technologies (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Biotin azide, by Vector Laboratories (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Sureselectqxt, by Agilent Technologies (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Ampure bead, by Beckman Coulter (1 mentions)
2100 bioanalyser, by Agilent Technologies (1 mentions)
C di amp, by Biolog (1 mentions)
Biotin c di gmp, by Biolog (1 mentions)
Cgamp, by Biolog (1 mentions)
Tnt t7 coupled wheat germ extract system, by Promega (1 mentions)
28 protocols using streptavidin coated beads
1
Biotin-labeled miR-127-5p Pulldown Assay
2
DOR DNA-DnaA/B/C Interaction Assay
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This assay was performed as previously described with minor modifications (7 , 8 (link)). DOR fragments were amplified by PCR, using pBSoriC (for Left-DOR, Middle/Right-DOR, and Right-DOR) and pBSoriCR4Tma (for Middle/Right-DOR R4Tma) as templates and primers listed in Table S3 . Biotinylated DOR DNA fragments (250 fmol) were incubated for 10 min at 4 °C in pull-down buffer (20 mM Hepes–KOH at pH 7.6, 1 mM EDTA, 4 mM DTT, 5 mM magnesium acetate, 40 mM ammonium sulfate, 20 mM NaCl, 10% [v/v] glycerol, 0.1% TritonX-100, 1 mM ATP, and 0.1 mg/ml BSA), containing DnaA (10 pmol), His-DnaB (10 pmol), and DnaC (10 pmol). Biotinylated DNA-bound materials were recovered using streptavidin-coated beads (Invitrogen), washed in pull-down buffer (25 μl) excluding BSA, dissolved in SDS sample buffer, and analyzed by SDS–11% PAGE and silver staining. In parallel, quantitative control DNA was used for ethanol precipitation and electrophoresis. Sixty percent of the input DNA (about 150 fmol) was recovered in the bead-bound fraction in the experiments.
3
Isolation and Analysis of circPTCD3
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The biotin-labelled circPTCD3 and miR-198 probe and their control probes were synthesized by Sangon Biotech (Shanghai, China). In addition, the probe-coated beads were generated through the co-incubation of the probe with the streptavidin-coated beads (Invitrogen, CA, USA) at 25°C for 2 h. Cells were gathered and incubated with circPTCD3 probes overnight at 4°C. Following that, the beads were eluted and the complex was purified with TRIzol (Takara, Dalian, China). Afterwards, the abundance of both circPTCD3 and miR-198 was analyzed by the qPCR.
4
Quantitative Analysis of AAV5 Capsids
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SMC was performed on the Singulex® Erenna® instrument (MilliPore Sigma). Biotinylated anti-AAV5 capture antibodies (Progen, Cat no. 615148) and proprietary Erenna® labeled anti-AAV5 detection antibodies (ADK5b) were diluted in TBST and added to test samples, standards, and controls (diluted to MRD 4), Samples were subsequently captured using streptavidin-coated beads (Invitrogen catalog no. 65001) and a magnetic plate. After washing, proprietary elution buffer was added at 20% of the original sample volume. On the Erenna® instrument, samples were taken up by a narrow capillary where antibodies passed through an interrogation space for single-molecule counting using a fluorescent signal. Event Photons (EPs; low end and mid-level signal) were used for reporting and the signals measured were directly proportional to the amount of AAV5 capsids in the sample.
5
Biotin-labeled XIST Probe Purification
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Biotin-labelled XIST probes (sense and antisense) were synthesized by Sangon Biotech (Shanghai, China). Probe-coated beads were generated by incubating the probe with streptavidin-coated beads (Invitrogen) at 25°C for 2 h. A549 and H1299 cells were collected, lysed, and incubated with biotin-labelled probes overnight at 4°C. Then the beads were eluted and RNA-protein complexes purified with TRIzol (Takara, Dalian, China). SMAD2 expression was analyzed by western blot.
6
Affinity Capture of AK136714 Interactors
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The biotinylated probe of AK136714 and the control probe were synthesized by Sangon Biotech (Shanghai, China). Probe-coated beads were generated by co-incubating the probe with streptavidin-coated beads (Invitrogen, CA, USA) at 25° C for 2 h. Cells were lysed to extract total protein. After pretreatment of magnetic beads, RNA and beads were mixed. After separation, western blot and mass spectrometry were used to verify the downstream binding protein of AK136714.
7
Quantifying Active Caspase-2 in PC12 Cells
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We adapted an unbiased caspase-2 activity measurement from Jean et al.43 (link) bVAD-fmk (biotin-Val-Ala-DL-Asp-fluoromethylketone; 50 μM, MP Biomedicals), a biotinylated pan-caspase inhibitor that traps active caspases, was added to PC12 cultures 1 hr prior to treatment with Pen1-CRADD. Cells were lysed in CHAPS buffer. Active caspase-bVAD-fmk complexes were pulled out with streptavidin-coated beads (Invitrogen). Active caspase-2 was detected by western blotting using affinity-purified polyclonal anti-caspase-2 antibody.42 (link)
8
Exome sequencing for variant identification
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Exome sequencing was performed on patient and both parents using the Agilent SureSelectXT HumanAllExon V4 (50 Mb) (Agilent Technologies). Briefly, 3 μg of genomic DNA was sheared in 130 μl of low TE buffer to a peak size of 150–200 bp using Covaris E220, and then purified with AmpPure XP beads to remove fragments less than 100 bp. The purified DNA fragments were then subjected to Agilent SureSelect Library preparation Kit, ILM, to be end-repaired, A-tailed, and ligated to indexing-specific paired-end adaptor. The adapter-ligated libraries were amplified for five cycles using Herculase II (Agilent Technologies). Amplified Pre-capture libraries (750 ng) were concentrated in 3 μl and hybridized to the target specific baits (SureSelectXT Human All Exon V4; Agilent Technologies) according to the manufacturer’s recommendations. Hybridized material was captured using streptavidin-coated beads (Invitrogen, Paisley, UK) and amplified for 10 cycles. Captured libraries were pooled in pairs and paired-end sequenced on one lane of the Illumina HiSeq 2000 at the Stanford Center for Genomics and Personalized Medicine.
9
Identifying AK045171-Interacting Proteins
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A biotin-labelled AK045171 probe and control probe were synthesized by Sangon Biotech (Shanghai, China). Probe-coated beads were generated by co-incubation of the probe with streptavidin-coated beads (Invitrogen, CA, USA) at 25°C for 2 h. Cardiomyocytes were collected, lysed and incubated with the AK045171 probes overnight at 4°C. Thereafter, the beads were eluted, and the complex was purified with TRIzol (Takara, Dalian, China). Then, the abundance of SP1 was analysed via western blot.
10
Phospholipid-coated Magnetic Bead Preparation
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Phospholipid coated magnetic beads were prepared as previously described with minor modifications.31 Streptavidin coated beads (Invitrogen) were washed twice with 0.5% BSA in 20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl (TBS), 5 mmol/L CaCl2 and quenched with 2% BSA in TBS, 5 mmol/L CaCl2 for 4 hours at 37°C with mixing. Subsequently, the beads were mixed with 2 mmol/L phospholipid vesicles (DOPC/DOPS/DOPE/biotinylated DOPE; 60:20:18:2) at room temperature, overnight with rotation. Finally, the beads were washed twice and re‐suspended in 0.5% BSA in TBS, 5 mmol/L CaCl2 at 2.5 mg/mL.
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