Streptavidin coated Dynabeads (300 μL/sample, MyOne streptavidin C1, Thermo Fisher) were added to the PD10 column eluate and the samples were incubated spinning slowly on a wheel at 4°C for 3 h. The beads were separated from the supernatant using a Dyna-Mag magnet (Invitrogen) and washed extensively as described (Roux et al., 2012 (link)). Washed beads were frozen at -80°C before analysis by MS. Ten percent of the beads were taken and run on SDS-PAGE gels for western blot analysis. Beads were resuspended in SDS PAGE sample buffer containing 1 mM biotin and 10 mM DTT and heated at 95°C for 10 min before separation by SDS-PAGE.
Myone streptavidin c1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MyOne Streptavidin C1 is a magnetic bead-based product designed for the capture and purification of biotinylated molecules. It utilizes streptavidin-coated beads to bind to biotin-labeled targets, allowing for efficient separation and isolation. The product is suitable for a range of applications where the separation and purification of biotinylated compounds are required.
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Lab products found in correlation
Dynamag magnet, by Thermo Fisher Scientific (1 mentions)
Instantblue, by Abcam (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
96 well polystyrene plate, by Thermo Fisher Scientific (1 mentions)
Sulfuric acid h2so4, by Thermo Fisher Scientific (1 mentions)
Dynabeads his tag isolation and pulldown, by Thermo Fisher Scientific (1 mentions)
Direct q system, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
7 protocols using myone streptavidin c1
1
Affinity Purification and Western Blot
2
Aptamer Affinity Measurement Protocol
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For affinity measurement of all full-length aptamer sequences, each individual sequence was synthesized and purified by Integrated DNA Technologies (IDT) without modification. To prepare aptamer particles for the fluorescence binding assay, aptamers were coated on to forward primer-conjugated particles via PCR and converted to single-stranded aptamer as previously described29 (link).
For affinity measurement of aptamer core sequences which do not have primer regions, all individual sequences were synthesized by Integrated DNA Technologies (IDT) with a biotin conjugated to the 5′ end. To prepare aptamer particles for the fluorescence binding assay, 1 µM of each biotinylated aptamer was first incubated with 107 MyOne Streptavidin C1 (Thermofisher) particles in 100 µl PBSMCT buffer (DPBS with 2.5 mM MgCl2, 0.9 mM CaCl2, 0.01% Tween 20) for 30 min at room temperature. The aptamer particles were then washed in PBSMCT via a magnetic separator to remove any unbound aptamer and then resuspended in 100 µl PBSMCT.
For affinity measurement of aptamer core sequences which do not have primer regions, all individual sequences were synthesized by Integrated DNA Technologies (IDT) with a biotin conjugated to the 5′ end. To prepare aptamer particles for the fluorescence binding assay, 1 µM of each biotinylated aptamer was first incubated with 107 MyOne Streptavidin C1 (Thermofisher) particles in 100 µl PBSMCT buffer (DPBS with 2.5 mM MgCl2, 0.9 mM CaCl2, 0.01% Tween 20) for 30 min at room temperature. The aptamer particles were then washed in PBSMCT via a magnetic separator to remove any unbound aptamer and then resuspended in 100 µl PBSMCT.
3
Microarray Fabrication for Single-Cell Analysis
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To prepare the VFAC, a 1-mm square chip with an array of microchambers was fabricated by the injection molding (Fluidware Technologies, Inc. (Kasukabe-shi, Saitama-ken, Japan) of polydimethylpolysiloxane (PDMS). In the array, 75-μm diameter microchambers with a depth of 70 μm were aligned in square grids at 125-μm intervals. Laser ablation processing (L. P. S. Works Co. Ltd. (Ohta-ku, Tokyo, Japan)) was performed to produce through-holes with diameters of 5–7 μm in the bottoms of the microchambers for cell capture. DNA probes with various types of cell tags were immobilized on magnetic beads (Thermo Fisher Scientific, MyOne Streptavidin C1). An inkjet printer designed for biological applications (Microjet Corp. LabJet 500) was used to inject 9.6 nl of bead solution containing 1% glycerol, 1–2 × 105 beads and 10 mM Tris HCl (pH 7.5) into each microchamber. The solvent of the injected solution was absorbed by a hydrophilic aluminum oxide porous membrane filter (GE Healthcare, Anopore (0.2-μm diameter pores)) at the bottom of the microchambers. The printer injected separately prepared bead solutions with different cell ID tags into predetermined microchambers such that the VFAC contained 100 microchambers with 100 cell-ID tags. Each microchamber also featured a porous surface with 5–10 × 109 high-density mRNA trapping probes per chamber.
4
Centrosome Purification and Mass Spectrometry
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To prepare samples for mass spectrometry, cells were treated as described above, but were additionally incubated with 50 µM biotin (Sigma, USA) for 24 h before harvesting. After centrosomes were purified, all fractions from the corresponding cell lines were combined and diluted with three volumes of lysis buffer plus 0.1% SDS. The solution was sonified using a microtip with the following settings: 1 min time, 10 s on, 20 s off, 45% power. The sonified centrosomes were incubated with magnetic streptavidin beads (MyOne Streptavidin C1; Thermo Fisher Scientific, USA) overnight. Washing was performed one time each in the following order: lysis buffer, SDS buffer (2% SDS in H2O), salt buffer (500 mM NaCl, 1% Triton X-100, 1 mM EDTA and 50 mM HEPES, pH 7.6), Tris buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.6). Purified proteins were eluted from the streptavidin beads with Tris buffer plus 5 mM biotin. The whole samples were run on a NuPAGE SDS gel and stained with InstantBlue (Expedeon, UK), and each lane was cut into ten equally sized pieces, which were send for mass spectrometric analysis as described previously (Zhang et al., 2016 (link)).
5
Proximity Biotinylation of PPP2R5C Substrates
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Potential PPP2R5C substrates were identified by proximity biotinylation using the BioID method described in [33 (link)]. PPP2R5C was fused to a mutated biotin ligase (BirA R118G) either to its C terminal (Myc-BirA-PPP2R5C) or N terminal ends (PPP2R5C-BirA-HA). These were co-transfected with or without a catalytic dead version (D85N) of the PP2A catalytic C subunit [34 (link)]. After 24 hours of expression, cells were washed with PBS twice and lysed in BioID lysis buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 0.4% SDS, 5 mM EDTA, 1 mM DTT, 2% Triton X-100, and 1x Complete protease inhibitor (Roche)). The lysates were mixed with an equal volume of 50mM Tris (pH 7.4) before incubating with pre-washed streptavidin magnetic beads (Invitrogen MyOne Streptavidin C1) overnight at 4°C. Then the beads were washed twice with 2% SDS, once with BioID wash buffer 1 (0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM Hepes, pH 7.5), once with BioID wash buffer 2 (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8.1), and twice with wash buffer 3 (50 mM Tris, pH 7.4, and 50 mM NaCl). Finally, all biotinylated proteins were eluted in 1x Laemmli buffer with saturated biotin (around 1mM) at room temperature for 15min and following 15 min boiling at 95°C. Eluted proteins were probed by immunoblotting.
6
EN2 Protein Expression and Purification
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Genomic DNA of EN2 was purchased from Thermo Fisher Scientific (USA). The pET28a plasmid was purchased from Novagen (Germany). Nucleospin Gel and PCR Clean-up/Plasmid EasyPure kit were acquired from Macherey–Nagel (Germany). E. coli strain Rosetta (DEC), Dynabeads his-tag Isolation and Pulldown, MyOne Streptavidin C1, and TOPO TA cloning kit were purchased from Invitrogen (USA). HisTrap Ni–NTA affinity column and Sephadex G-25 Superfine resin were purchased from GE Healthcare (USA). All DNAs were synthesized from Bionics Inc. (South Korea). For ELONA, EN2 monoclonal antibody was purchased from Abnova (Taiwan). Streptavidin Poly-HRP (poly-HRP), 3,3′,5,5′-tetramethylbenzidine (TMB) substrate kit, 96-well polystyrene plates, and sulfuric acid (H2SO4) were purchased form Thermo Fisher Scientific (USA). Bovine serum albumin (BSA) and IgG were purchased from Sigma-Aldrich (USA). Prostate specific antigen (PSA) and cancer antigen 125 (CA125) were purchased from Sino Biological Inc. (USA) and Abnova (Taiwan). Artificial urine medium (AUM) was purchased form Pickering Laboratories Inc. (USA). All materials were of analytical grade. All aqueous solutions were prepared in deionized water (> 18 MΩ) obtained using a Direct-Q system from Merck Millipore (USA).
7
Metabolic Labeling and Enrichment of Newly Synthesized Proteins
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4T1-mock and 4T1-METTL7A1 cells were labelled for 210 min with l -azidohomoalanine (AHA, Invitrogen). Extracts were prepared and Biotin Click-iT® reactions were performed exactly as described in Manufacturer's instructions. Then ‘Clicked’ reactions were diluted and precipitated (overnight at 4°C rotating) with Streptavidin beads (MyONE-Streptavidin C1, Invitrogen). After extensive washes, proteins were eluted from beads with 1× SDS-Sample Buffer and analyzed by SDS-PAGE and Immunoblotting.
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