Streptavidin beads

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

Streptavidin beads are a type of solid-phase affinity matrix used for the separation and purification of biotinylated molecules. They consist of streptavidin, a protein derived from the bacterium Streptomyces avidinii, which binds to biotin with high affinity. These beads provide a convenient platform for the capture, separation, and immobilization of biotin-labeled proteins, nucleic acids, and other biomolecules.

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263 protocols using streptavidin beads

Pull-down Assay for miR-181a-5p Regulation

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Pull-down assays were performed as described previously [27 (link)]. Briefly, S1-CRNDE and S1-CRNDE mutant were generated, and cotransfected with or without miR-181a-5p inhibitor. After 48 h of transfection, cells were harvested and washed by PBS for two times, then crosslinked in 0.37% formaldehyde, incubated in ice-cold lysis buffer (150 mM NaCl, 10 mM Hepes, 3 mM MgCl₂, 10% glyceral, 1% NP-40, 2 mM DTT, 1 mM PMSF, 1 × protenase inhibitor (Sigma), 10 ul RNase inhibitor (promega)). The cell lysate was precleaned in agarose beads (Santa Cruz Biotechnology) at 4 °C for 1 h, then incubated and rotated in streptavidin beads (Thermo Fisher Scientific, San Jose, CA, USA) at 4 °C for 3 h. The streptavidin beads were collected, washed in elution buffer (50 mM Hepes, 5 mM EDTA, 100 mM NaCl, 1% SDS, 10 mM DTT). The beads were heated at 70 °C for 45 min. miR-181a-5p expression was examined by qRT-PCR.

Han P., Li J.W., Zhang B.M., Lv J.C., Li Y.M., Gu X.Y., Yu Z.W., Jia Y.H., Bai X.F., Li L., Liu Y.L, & Cui B.B. (2017). The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling. Molecular Cancer, 16, 9.

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Identifying Protein Targets of Biotinylated Probes

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To identify the target protein for FB, FB-based biotinylated inactive probe 1 and active probe 2 were synthesized as described below. HeLa cell pellets were purchased from National Cell Culture Center and lysed in 20 ml of the NP-40 lysis buffer as indicated above. The cell lysates (250 mg protein) were pre-cleared with 200 μl streptavidin beads (Thermo Fisher Scientific) at 4°C for 1 h. Binding reactions were performed by incubating the pre-cleared cell lysates (125 mg proteins/40 ml) with 2 μM probe 1 or probe 2 at 4°C for 3 h, followed by adding 100 μl streptavidin beads and incubating the mixtures overnight at 4°C. After incubation, the beads were washed four times with the lysis buffer, and the bead-bound proteins were eluted, followed by digestion, LC-MS/MS analysis and MASCOT protein identification as previously reported (Yang et al., 2014 (link)). A decoy database was built and searched to determine the false discovery rates (FDR) and peptides with FDR lower than 0.01 were assigned as high confidence identification. The probability-based MOWSE scores calculated by MASCOT were used to determine the predominant protein in the sample.

Ye Q., Zhang Y., Cao Y., Wang X., Guo Y., Chen J., Horn J., Ponomareva L.V., Chaiswing L., Shaaban K.A., Wei Q., Anderson B.D., St Clair D., Zhu H., Leggas M., Thomon J.S, & She Q.B. (2019). Frenolicin B targets peroxiredoxin 1 and glutaredoxin 3 to trigger ROS/4E-BPl-mediated antitumor effects. Cell chemical biology, 26(3), 366-377.e12.

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Coprecipitation of Biotinylated Compounds

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For coprecipitation from culture medium, 7WD10 cells in 12-well plates were cultured for 48 h at 37 °C, and the culture medium were recovered. This culture medium was treated with biotinylated LME-tet (50 µM) or LME-mono (200 µM) for 24 h at 4 °C, and then treated with streptavidin beads (Thermo Fisher Scientific) for 24 h at 4 °C. After extensive washing, the beads were analyzed by western blot, as described above. For coprecipitation from cell lysates, 7WD10 cells in 12-well plates were cultured for 30 min at 4 °C or 37 °C in the presence or absence of biotinylated LME-tet (50 µM) or LME-mono (200 µM). The cells were recovered and lysed in lysis buffer, as described above. Lysates were treated with streptavidin beads (Thermo Fisher Scientific) for 24 h at 4 °C, and after extensive washing, the beads were analyzed by western blot, as described above.

Sato W., Watanabe-Takahashi M., Murata T., Utsunomiya-Tate N., Motoyama J., Anzai M., Ishihara S., Nishioka N., Uchiyama H., Togashi J., Nishihara S., Kawasaki K., Saito T., Saido T.C., Funamoto S, & Nishikawa K. (2023). A tailored tetravalent peptide displays dual functions to inhibit amyloid β production and aggregation. Communications Biology, 6, 383.

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Isolating EpCAM+ and HLA-G+ Cells from Blood

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For each blood sample 2 antibody-bead complexes were prepared. 17.5 μl of biotinylated anti-EpCAM antibodies (R&D Systems, BAF960) with a concentration of 0.2 mg/ml was incubated with 14 μl of streptavidin beads (Thermo Fisher Scientific, 65601) with a concentration of 10 mg/ml, and suspended in 140 μl of PBS. Also, 2 μl biotinylated anti-HLAG antibodies (Thermo Fisher Scientific, MA1-10361) with a concentration of 1 mg/mL was incubated with 8 μl of streptavidin beads (Thermo Fisher Scientific, 65601) with a concentration of 10 mg/ml, and suspended in 80 μl of PBS. Both incubations took 1 hour and were followed by a triple wash with PBS to remove any unconjugated antibodies.
The antibody-bead mixtures were incubated with 8 ml of blood for 35 mins. Afterwards the sample was diluted to 16 ml by adding 8 ml of PBS, and was run through the porous chip. The suspension was run though the porous chip 2 times, once using a flow rate of 2 ml/min and then again using 1 ml/min. The high volumetric throughput capability of the system facilitated the recirculation. This was then followed by a 4 ml PBS wash using a 1ml/min flow rate. After the wash, any remaining red blood cells were removed by a 10 minute incubation with a RBC Lysis buffer (Biosciences, 786–672) followed by a 2 min PBS wash.

Gur O., Chang C.L., Jain R., Zhong Y, & Savran C.A. (2020). High-purity isolation of rare single cells from blood using a tiered microchip system. PLoS ONE, 15(3), e0229949.

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Identification of ROCKI-interacting Proteins

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ROCKI, antisense of ROCKI, or lacZ control was cloned into pBluescript KSII downstream of the T7 promoter separately (primers listed in Table EV2). The vectors were linearized by KpnI digestion, and 1 μg of linear lacZvectors was transcribed and biotinylated in vitro using a kit (Epicentre), according to the manufacturer's instructions. Biotinylated RNAs were purified, refolded, and incubated with nuclear lysates from THP1‐derived macrophages using the method described previously (Li et al, 2014). Briefly, 2 μg of diluted RNA (200 ng/μl) was incubated at 60°C for 10 min and slowly cooled to 4°C. Nuclear lysates were prepared from 2 × 10⁸ PMA‐differentiated THP1‐derived macrophages treated with 100 ng/ml Pam3 for 8 h. Protein concentrations were measured by the DC assay (Bio‐Rad) according to the manufacturer's instructions. For the pull‐down incubations, nuclear lysates (1 mg of protein) were precleared with streptavidin beads (Invitrogen) and then incubated with 2 μg of biotinylated RNA and 40 μl of streptavidin beads for 2 h at 4°C. The beads were collected and washed three times with buffer C. RNA‐associated proteins were eluted and analyzed by mass spectrometry (Sanford Burnham Prebys Medical Discovery Institute Core) or by Western blotting, as described below.

Zhang Q., Chao T., Patil V.S., Qin Y., Tiwari S.K., Chiou J., Dobin A., Tsai C., Li Z., Dang J., Gupta S., Urdahl K., Nizet V., Gingeras T.R., Gaulton K.J, & Rana T.M. (2019). The long noncoding RNA ROCKI regulates inflammatory gene expression. The EMBO Journal, 38(8), e100041.

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RNA immunoprecipitation and circRNA pulldown

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For the RNA immunoprecipitation assay, the EZMagna RIP kit (Millipore, USA) was used, following the manufacturer’s protocol. For the circRNA pull-down assay, biotin-labeled circZFR probes were synthesized by RiboBio and the assay was performed as described previously. [15 ] Briefly, cell lysates were prepared in the IP lysis buffer and pre-cleared by incubation with streptavidin beads (65,001, Invitrogen) at 4 °C for 1 h. CircRNA probes immobilized on the streptavidin beads were then added to the cell lysates and incubated overnight at 4 °C. After washing five times, the beads were boiled in SDS buffer for protein elution and MS.

Ma Z., Chen H., Xia Z., You J., Han C., Wang S., Xia W., Bai Y., Liu T., Xu L., Zhou G., Xu Y, & Yin R. (2023). Energy stress-induced circZFR enhances oxidative phosphorylation in lung adenocarcinoma via regulating alternative splicing. Journal of Experimental & Clinical Cancer Research : CR, 42, 169.

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Identifying LINC00886-Interacting Proteins

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Initially, LINC00886 and antisense-LINC00886 RNAs were labelled by the Biotin RNA Labeling Mix (Roche, USA) and puri ed with an RNeasy Mini Kit (QIAGEN, USA). Then, the biotinylated RNAs were incubated with streptavidin beads (Invitrogen, USA), and the streptavidin beads combined with and without RNAs were mixed with protein extracts of CAL-62 cells at 4 °C on a rotator overnight. The beads were washed gently three times in 1 × washing buffer (5 mM Tris-HCl, 1M NaCl, 0.5 mM EDTA, and 0.005% Tween 20), followed by mix with DEPC and 5 × SDS buffer. The proteins were separated by gel electrophoresis and visualized by silver staining. Speci c bands were identi ed by mass spectrometry analysis and retrieved in the human RefSeq protein database (National Center for Biotechnology Information), using Mascot version 2.4.01 (Matrix Science, London, UK). The retrieved protein was detected by western blot. The primers of LINC00886 are provided in Table S1.

Ma B., Luo Y., Xu W., Han L., Liu W., Jiang H., Wang X., Wen S., Liao T., Wei W., Ji Q., & Wang Y. (2020). LINC00886, as a Biomarker Indicative of Thyroid Cancer Dedifferentiation, Negatively Regulates the Malignancy in Anaplastic Thyroid Cancer.

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Quantifying Protein Sulfenylation via DCP-Bio1

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To measure sulfenic acid (CysOH) formation (sulfenylation) of proteins, cells were lysed in degassed-specific lysis buffer [50 mM HEPES, pH7.0 at room temperature, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 10 mM sodium pyrophosphate, 5 mM Iodoacetamide (IAA), 100 μM DTPA, 1% Triton-X-100, protease inhibitor, 200 unit/mL catalase (Calbiochem), 200 μM DCP-Bio1 (KaraFast, USA)] and then DCP-Bio1-bound proteins were pulled down with streptavidin beads (Thermo scientific, USA) overnight at 4 °C. DCP-Bio1-bound CysOH formed, sulfenylated-proteins were determined by immunoblotting with specific antibodies, as reported ^{19 (link), 40 (link), 61 (link)}.

Das A., Ash D., Fouda A.Y., Sudhahar V., Kim Y.M., Hou Y., Hudson F.Z., Stansfield B.K., Caldwell R.B., McMenamin M., Littlejohn R., Su H., Regan M.R., Merrill B.J., Poole L.B., Kaplan J.H., Fukai T, & Ushio-Fukai M. (2022). Cysteine Oxidation of Copper transporter SLC31A1/CTR1, drives VEGFR2 signaling and Angiogenesis. Nature cell biology, 24(1), 35-50.

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RNA-Protein Interaction Identification

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Probe-ATB or probe-control was transcribed from ATB shRNA lentivector and labeled in vitro by Biotin RNA Labeling Mix (Roche, Basel, Switzerland) to carry out RNA pull-down assay. Secondary structure was formed through biotin-labeled RNAs by adopting the RNA structure buffer (Thermo Fisher Scientific, MA, United States). Then, RNA immunoprecipitation wash buffer (500 μl, Thermo Fisher Scientific) was utilized to rinse Streptavidin beads (Thermo Fisher Scientific) thrice, while the beads were later mixed with biotinylated RNAs overnight under 4°C. Later, the magnetic field was applied to separate the overnight mixture to obtain the streptavidin bead–RNA complexes. Afterward, complexes were mixed with cell lysates before 1 h of incubation on the rotator under ambient temperature. Again, the magnetic field was applied to separate the mixture after incubation for obtaining streptavidin bead–RNA–protein complexes.

Mi Y.Y., Sun C.Y., Zhang L.F., Wang J., Shao H.B., Qin F., Xia G.W, & Zhu L.J. (2021). Long Non-coding RNAs LINC01679 as a Competitive Endogenous RNAs Inhibits the Development and Progression of Prostate Cancer via Regulating the miR-3150a-3p/SLC17A9 Axis. Frontiers in Cell and Developmental Biology, 9, 737812.

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Measuring VE-Cadherin Protein Turnover

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COS7 cells were transfected with the plasmid of interest 48 h before to the experiment. Cells were pulse-labeled with biotin (21331; ThermoFisher) on ice for 30 min. Excess biotin was quenched by 50 mM ammonium chloride. Cells were then incubated at 37°C for various amounts of time, returned to ice, and then lysed with 1% Triton X-100 in PBS with calcium and magnesium for 30 min. Cell lysates were centrifuged at 13,200 rpm for 30 min at 4°C. The supernatants were added to streptavidin beads (20349; ThermoFisher) and incubated for 1 h at 4°C. Beads were washed four times using ice-cold PBS with 0.1% Tween-20. biotinylated protein was eluted using Laemmli Sample Buffer with 5% β-ME at 95°C for 5 min, followed by Western blot analysis.
The following equation was used calculate VE-cadherin half-life:

with the rate constant

where t_1/2 is the half-life, t is the given time, N_t is the amount of protein at the given time, and N₀ is the amount of protein at time 0.

Su W, & Kowalczyk A.P. (2017). The VE-cadherin cytoplasmic domain undergoes proteolytic processing during endocytosis. Molecular Biology of the Cell, 28(1), 76-84.

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