Dynabeads myone streptavidin c1

Manufactured by Thermo Fisher Scientific

Sourced in United States

Dynabeads MyOne Streptavidin C1 are uniform, superparamagnetic polystyrene beads coated with streptavidin. They are designed for the efficient isolation and purification of biotinylated molecules, including proteins, nucleic acids, and cells.

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Lab products found in correlation

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278 protocols using dynabeads myone streptavidin c1

Biotinylated Protein Purification for Proteomic Analysis

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Biotinylated proteins from BioID experiments were purified for proteomic analysis with Dynabeads MyOne Streptavidin C1 (Invitrogen) as previously described [32 (link)]. Briefly, cell lysates in RIPA buffer (500 μg) were incubated overnight at 4°C with 50 μL slurry of Dynabeads MyOne Streptavidin C1 (Invitrogen) and then washed for 10 min on a rotator at room temperature as follows: twice with washing buffer 1 (2% SDS in ddH₂O), twice with washing buffer 2 (0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM HEPES, pH 7.5), twice with washing buffer 3 (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8.1) and twice with washing buffer 4 (50 mM Tris, pH 7.4, and 50 mM NaCl). Biotinylated proteins were dissociated from beads by incubation with 25 μL elution buffer (10 mM Tris, pH 7.4, 2% SDS, 5% (v/v) β-mercaptoethanol and 2 mM biotin) for 15 min at 98°C.

Porcellato E., González-Sánchez J.C., Ahlmann-Eltze C., Elsakka M.A., Shapira I., Fritsch J., Navarro J.A., Anders S., Russell R.B., Wieland F.T, & Metzendorf C. (2022). The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes. PLoS ONE, 17(8), e0261543.

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Affinity Purification of Biotinylated Proteins

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Cells were incubated in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM CaCl₂, 1 mM MgCl₂) supplemented with the protease inhibitor cocktail (Sigma-Aldrich) on ice for 1 h and centrifuged at 17000 × g (4°C) for 15 min. Protein concentration was estimated using BCA Kit (Pierce). To avoid unspecific binding, cell lysates were precleared using Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen). 70 μg of biotinylated HPA was incubated with 0.3 mg of the total cell lysate proteins in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM CaCl₂, 1 mM MgCl₂ supplemented with protease inhibitor cocktail for 3 h at 4°C. After this time, 100 μl Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen) was added to the mixture for 1 h at 4°C. Finally, the beads were washed 4 × 1 ml of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM CaCl₂, 1 mM MgCl_2, after which proteins were harvested by adding 2× Laemmli buffer and incubating the beads at 96°C for 5 minutes. Samples were resolved in 10% acrylamide gel followed by silver staining using Silver Staining Kit (Pierce) or probed with a specific antibody in the western blotting assay.

Khosrowabadi E., Wenta T., Keskitalo S., Manninen A, & Kellokumpu S. (2022). Altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc defines the highly invasive cancer cell phenotype. Oncotarget, 13, 73-89.

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Peptide Pull-Down Assay for Integrin Interactors

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Pull-down assays were performed with N-terminally desthio biotinylated β1-WT, β1-Scr, β1-TT/AA, β1-TT/DD, β1-pTpT peptides. Before use, peptides were immobilized on 25 μl Dynabeads MyOne Streptavidin C1 (10 mg/ml; Invitrogen) in Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) containing protease inhibitors (cOmplete; Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 and 3; Sigma-Aldrich) for 3 h at 4°C. Cell lysates were prepared with MPER containing protease and phosphatase inhibitors and 0.5 mg of supernatant was incubated with the peptide-coated beads at 4°C under constant rotation. For pull-downs with recombinant proteins, synthetic β1 integrin tail peptides were immobilized on 25 μl Dynabeads MyOne Streptavidin C1 (10 mg/ml; Invitrogen) for 3 h at 4°C, incubated with 2% BSA in Mammalian Protein Extraction Reagent (Thermo Scientific Scientific) for 30 min at 4°C to block unspecific binding before adding kindlin-2 or THD (conc 2.5 and 8.5 nM, respectively) and further incubation on a rotator for 2 h at 4°C. After washing the beads three times with lysis buffer, the proteins bound to the beads were eluted using Laemmli buffer, separated on a 10% SDS–PAGE, and analyzed by Western blotting.

Böttcher R.T., Strohmeyer N., Aretz J, & Fässler R. (2022). New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain. Life Science Alliance, 5(4), e202101301.

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Aptamer-based DEHP Detection Assay

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Both 1,2,4-benzenetricarboxylic acid 1,2-bis(2-ethylhexyl) ester and bis(2-ethylhexyl)phthalate (DEHP) were purchased from Toronto Research Chemicals (ON, Canada). N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) were purchased from CovaChem (IL, USA). Magnetic beads (Dynabeads MyOne Streptavidin C1) were purchased from Thermo Fisher Scientific (CA, USA). Proprietary bis(2-ethylhexyl)phthalate (DEHP) aptamers were purchased from Base Pair Biotechnologies (TX, USA). A biotin and an 18-atom hexa-ethyleneglycol spacer were added at the 5′-end of the aptamer. Hydroxylamine hydrochloride, sodium hydroxide, silver nitrate, tetraethyl orthosilicate (TEOS), (3-aminopropyl) trimethoxysilane (APTMS), ammonium hydroxide (28%), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), sodium chloride (NaCl), sodium phosphate, ethanol, dimethyl sulfoxide (DMSO), and isopropanol were purchased from Sigma Aldrich (MO, USA). Milli-Q ultrapure water (18.2 MΩ cm⁻¹) was used in all the procedures. The mineral water and carbonated drinks in polyethylene terephthalate (PET) bottles were purchased from a local supermarket (TX, USA).

Tu D., Garza J.T, & Coté G.L. (2019). A SERS aptasensor for sensitive and selective detection of bis(2-ethylhexyl)phthalate. RSC Advances, 9(5), 2618-2625.

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Enrichment and Identification of 8-oxoG in DNA

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DNA fragments harboring 8-oxoG were enriched as previously described^{37 (link)}, with minor modifications. Briefly, 5 µg of the genomic DNA extracted from adipose or lung tissues of randomly selected mice (n = 2) were sheared with an S2 ultrasonicator (Covaris, Woburn, MA, USA) in 10 mM Tris buffer (pH 8.0) to obtain ~150-bp fragments. After sonication, the fragmented DNA was concentrated to 20 µl in 100 mM NaPi buffer (pH 8.0) using QIAquick PCR purification kit (Qiagen). A 100-µl volume of 100 mM NaPi buffer containing 20 mM amine-PEG2-biotin (Thermo Fisher Scientific) was added and the mixture was heated to 75 °C for 10 min. After thermal equilibration, 5 mM K₂IrBr₆, a mild one electron oxidant, was added for 1 h for 8-oxoG biotinylation through covalent adduction. The DNA fragments biotinylated at 8-oxoG were eluted with 125 µl Tris buffer using the QIAquick PCR purification kit and extracted using Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific); strands complementary to those with bound biotinylated 8-oxoG were released by incubation in 150 mM NaOH at 20 °C for 30 min, and concentrated to 10 µl ddH₂O using ssDNA/RNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA).

Park J.W., Han Y.I., Kim S.W., Kim T.M., Yeom S.C., Kang J, & Park J. (2019). 8-OxoG in GC-rich Sp1 binding sites enhances gene transcription in adipose tissue of juvenile mice. Scientific Reports, 9, 15618.

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In Vitro RNA and DNA Editing Assays

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The in vitro editing reaction mixture, containing 5 nM of telomere RNA:RNA duplex substrates and 75 nM of HAT-ADAR1p110-WT, FLAG-ADAR1p110-WT, or HA-ADAR1p110-EAA protein, was incubated at 37 °C for 2 h in in vitro editing buffer I (20 mM HEPES-KOH pH 7.5, 100 mM NaCl, 0.01% NP-40, 5% glycerol, 1 mM DTT). For editing of RNA:DNA hybrid substrates, in vitro editing buffer II (20 mM HEPES-KOH pH 7.5, 20 mM NaCl, 0.01% NP-40, 5% glycerol, 1 mM DTT) was used. Edited RNA or DNA strands were purified using Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). To remove opposite RNA or DNA strands, RNase H (NEB) or TURBO DNase (Thermo Fisher Scientific) was used, respectively. For sequencing of edited substrates, reverse transcription-PCR was carried out for RNA strands, while PCR was carried out for DNA strands. Each reaction used specific primer sets (Supplementary Data 1). RT reactions were carried out using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and PCR reactions were performed using Platinum Taq DNA polymerase (Thermo Fisher Scientific). PCR products were sequenced using a specific sequencing primer, and the ratio of A and G peaks in the chromatograms were analyzed by CodonCode Aligner (CodonCode Corporation).

Shiromoto Y., Sakurai M., Minakuchi M., Ariyoshi K, & Nishikura K. (2021). ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells. Nature Communications, 12, 1654.

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Nascent RNA-Seq Analysis of Transcriptional Elongation

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~20 million cells were treated with 250 nM NVP-2 for 1 h, followed by nascent RNA labeling with 500 µM 4-thiouridin (4sU) (Sigma # T4509) for 15 min. TT-seq library preparation was performed as described previously (Rosencrance et al., 2020 (link); Schwalb et al., 2016 (link)). Briefly, following 4sU labeling, total RNA was extracted using TRIzol (ThermoFisher # 15596026). Spike-in was not included, since this experiment was shown to quantify elongation rates and not Pol II levels, which were already shown by the ChIP-seq and PRO-seq. Total RNA was fragmented using Magnesium RNA Fragmentation Module (NEB #E6150S), followed by biotinylation of 4sU using Biotin-XX-MTSEA (Biotium #90066) in 20% DMF. Biotinylated RNA was enriched using Dynabeads MyOne Streptavidin C1 (ThermoFisher #65001), followed by reduction of disulfide bonds in 100 mM DTT for RNA elution. DNA libraries that were prepared from the enriched RNA fragments were sequenced on NovaSeq 6000. Low-quality bases were removed from 3’ ends using cutadapt 1.14 (Martin, 2011 ). Reads were aligned to human hg38 genome assembly with Ensembl annotation data release 103 using STAR 2.7.5 (Dobin et al., 2013 (link)). Counts of reads with primary alignments and MAPQ ≥ 7 were normalized to total mapped reads.

Aoi Y., Takahashi Y.H., Shah A.P., Iwanaszko M., Rendleman E.J., Khan N.H., Cho B.K., Ah Goo Y., Ganesan S., Kelleher N.L, & Shilatifard A. (2021). SPT5 stabilization of promoter-proximal RNA polymerase II. Molecular cell, 81(21), 4413-4424.e5.

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Phendione Derivative Clickable Proteome

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Phendione derivative (Alk-Phen) (structure shown in Supplemental Fig. S4) was custom-synthesized. Cells were treated with 5 µM Alk-Phen, 3 µM phendione, or DMSO for 3 h. Whole-cell lysate was obtained using lysis buffer containing 150 mM NaCl, 1.0% NP-40, and 50 mM Tris-HCl (pH 8.0) and dialyzed against buffer containing 10 mM NaCl, 1.0% NP-40, and 50 mM Tris-HCl (pH 8.0). The cell lysate was then clicked to biotin by adding 50 μM biotin picolyl azide (Click Chemistry Tools 1167), a preformed complex of CuSO₄:THPTA (1.0 mM:5.0 mM), and 5 mM sodium ascorbate, sequentially. The click reaction was performed for 30 min at room temperature protected from light. The cell lysate was then dialyzed against lysis buffer to remove unclicked azidobiotin and pull-down was carried out with Dynabeads MyOne streptavidin C1 (Thermo Fisher Scientific 65002).

Yue J., Vendramin R., Liu F., Lopez O., Valencia M.G., Gomes Dos Santos H., Gaidosh G., Beckedorff F., Blumenthal E., Speroni L., Nimer S.D., Marine J.C, & Shiekhattar R. (2020). Targeted chemotherapy overcomes drug resistance in melanoma. Genes & Development, 34(9-10), 637-649.

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Single-Cell 3′ RNA Sequencing Protocol

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The 3′READS protocol (3′READS+ version) was used in this study as previously described (Zheng et al., 2016 (link)). Briefly, poly(A)⁺ RNA was captured with oligo d(T)₂₅ magnetic beads (NEB) and fragmented on-beads by RNase III. After washing away free RNA fragments, the poly(A)-containing RNA fragments were eluted from the beads and precipitated with ethanol and ligated to heat-denatured 5′adaptor (5′-CCUUGGCACCCGAGAAUUCCANNNN) with T4 RNA ligase 1 (NEB). The ligated products were then captured biotin-T₁₅-(+TT)₅(Exiqon), where +T is locked nucleic acid, bound to Dynabeads MyOne Streptavidin C1 (Thermo Fisher). Bound RNA was digested with RNase H, which also eluted RNA from the beads. Eluted RNA fragments were precipitated with ethanol after washing with RNase H buffer. Purified RNA fragments were then ligated to a 5′-adenylated 3′ adaptor (5′-rApp/NNNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC) with T4 RNA Ligase 2 (truncated KQ, NEB). The ligation products were reverse-transcribed M-MLV reverse transcriptase (Promega), followed by PCR amplification using Phusion high-fidelity DNA polymerase (NEB) and bar-coded PCR primers for 15–18 cycles. PCR products were size-selected twice with AMPure XP beads (Beckman Coulter). The size and quantity of the libraries were examined on an Agilent Bioanalyzer. The cDNA libraries were purified and sequenced on an Illumina HiSeq (1x150 nt).

Cheng L.C., Zheng D., Zhang Q., Guvenek A., Cheng H, & Tian B. (2021). Alternative 3′ UTRs play a widespread role in translation-independent mRNA association with the endoplasmic reticulum. Cell reports, 36(3), 109407.

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Quantification of hMPV Viral RNAs

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Confluent A549 cells in 150-mm dishes were mock infected or infected with WT or mutant rhMPV at an MOI of 0.5. At day 2 postinfection, total RNA was isolated from virus-infected cells using the TRizol reagent (Life Technologies) and dissolved in RNase-free water. Subsequently, poly(A)-containing RNA was isolated from total RNA using a Dynabeads mRNA Direct™ kit (Life Technologies) according to the manufacturer’s recommendations. Finally, hMPV G mRNA was isolated by Dynabeads MyOne™ Streptavidin C1 (ThermoFisher Scientific) conjugated with poly T-tailed G gene specific primer. Virion RNA was extracted from sucrose-gradient purified virions of rhMPV or rhMPV mutant. HMPV genome, antigenome, and G mRNA were quantified by real-time RT-PCR.

Lu M., Zhang Z., Xue M., Zhao B.S., Harder O., Li A., Liang X., Gao T.Z., Xu Y., Zhou J., Feng Z., Niewiesk S., Peeples M.E., He C, & Li J. (2020). N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I. Nature microbiology, 5(4), 584-598.

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