Purified recombinant IgGs were covalently coupled to MyOne Tosylactivated Dynabeads (Thermo Fisher Scientific, Cat#: 65501). Coupling and Env-immunoprecipitation were carried out according to the manufacturer’s protocol. Briefly, 5 mg of Env produced in HEK293S GnTI−/− were incubated with 200 μg of IgG-beads for 30 min. The IgG-Env protein complexes were then precipitated using magnetic separation and washed 3–4× before performing acidic elution and pH neutralization of the bound material. Env-samples of the original input of 426c WT Core or HxB2 WT Core, and bead-bound/eluted and unbound fractions were subjected to SDS gel electrophoresis under reducing conditions. A sample of the bound fractions were subjected to LC-MS/MS analysis or used for BLI.
Myone tosylactivated dynabeads
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Italy
MyOne Tosylactivated Dynabeads are high-performance magnetic beads designed for covalent coupling of proteins, peptides, or other ligands. They feature a uniform size distribution and superparamagnetic properties, allowing for efficient separation and purification of target molecules.
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9 protocols using myone tosylactivated dynabeads
1
IgG-based Env Immunoprecipitation Protocol
2
LPS-Induced Inflammation and VCAM-1 Imaging
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Lipopolysaccharide from Escherichia coli (clone 055:B5, Sigma-Aldrich, St. Louis, MO, United States) was dissolved to 1 mg/ml in sterile PBS and intraperitoneally administered to 4-month-old mice at a dose of 1 mg/kg. Two hours after LPS administration, mice were treated with 60 mg/kg CuII(atsm) dissolved in SSV solution containing 0.9% (w/v) NaCl, 0.5% (w/v) sodium carboxymethylcellulose, 0.5% (v/v) benzyl alcohol, and 0.4% (v/v) Tween 80, or SSV alone by oral gavage. CuII(atsm) was prepared according to published procedures (Gingras et al., 1962 (link)). 23.5 h after LPS administration, mice were injected with MPIO-VCAM-1 (conjugate) that was prepared according to Montagne et al. (2012) (link) by coating MyOne Tosyl Activated Dynabeads (Thermo Fisher Scientific, Waltham, MA, United States) with 0.1 M sodium borate buffer (pH 9.5) and conjugating with VCAM-1 antibody (40 μg/1 mg of beads, BD Biosciences, San Jose, CA, United States) in 3 M ammonium sulfate at 37°C for 48 h . The conjugate was blocked with 0.5% (w/v) bovine serum albumin (BSA) in PBS-Tween for 24 h at 37°C and stored in 0.1% (w/v) BSA in PBS-Tween at 4°C. Each animal received an intravenous dose of the MPIO-VCAM-1 conjugate containing 1.0 mg/kg of Fe 30 min prior to imaging.
3
IgG-based Env Immunoprecipitation Protocol
4
Immunoaffinity Isolation using Tosylactivated Dynabeads
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For immunoaffinity isolation, we used MyOne Tosylactivated Dynabeads (Life Technologies, Grand Island, NY; #65502), which are based on 1 µm superparamagnetic beads. Antibodies were coupled at 40 µg antibody per mg of beads in 0.1 M sodium borate pH 9.5, 1 M ammonium sulfate; coupling went overnight at 37°C with shaking. Unreacted groups were blocked overnight at 37°C with shaking in PBS containing 0.05% Tween 20, and 0.5% BSA. Antibody-coupled beads were stored at 4°C in the same buffer with 0.02% NaN3. The D10 bead stock concentration was 50 mg/ml, with the coupled antibody at 2 mg/ml.
5
Peptide-Mediated Separation for MAP
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The PMS was performed on 1 mL of each sample (prepared as indicated above) using 5 µL of biotinyl-ated-aMp3 peptide-and 5 µL of biotinylated-aMptD peptide-coated MyOne Tosylactivated Dynabeads (Life Technologies, Paisley, Scotland, UK), prepared in-house as previously described (Foddai et al., 2010) . A positive (1 mL of a 10 -1 dilution of a broth culture of MAP B2) and negative (1 mL of 7H9 broth) control sample was included with each batch of 15 to 20 CMR samples processed. Magnetic separation was carried out using a Dynal BeadRetriever (Life Technologies). The MAP cell capture was carried out for 30 min at room temperature under continuous mixing, followed by 2 washes in 1 mL of PBS-T, and final resuspension of the beads in 1 mL of Middlebrook 7H9 broth containing 10% (vol/vol) OADC supplement (7H9/OADC broth) and NOA antibiotic supplement (Abtek Biologicals Ltd., Liverpool, UK; final concentrations per liter: 50,000 IU of nystatin, 2 mg of oxacillin, and 30 mg of aztreonam). This final 1-mL bead sample was split equally between the phage assay and culture, carried out as described below. The positive and negative PMS controls were also processed through the phage assay (PMS-phage assay) and culture (PMS-culture) along with each batch of test samples.
6
Immunoprecipitation for Testicular Antigen
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Immunoprecipitation was performed as described elsewhere [7 (link)]. In brief, prior to the immunoprecipitation, Ts4 or control mouse IgM (each 80 μg) was conjugated with 2 mg of Dynabeads MyOne Tosylactivated (Invitrogen) according to manufacturer’s instructions. Total proteins (50 μg) of the TS testicular extract in buffer A containing 0.1% Triton X-100 was treated with the Ts4 or normal control IgM-conjugated Dynabeads on a rotary shaker at 4°C overnight. After washing with PBS three times, the beads were boiled for 5 min in 3 × SDS buffer [9 (link)] under reducing conditions. After the centrifugation, the supernatants were used as samples for SDS-PAGE.
7
Antibody Conjugation to Magnetic Beads
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Borate buffer was prepared as follows: 3 g of boric acid (Sigma), 20 mL of water, 5 mL of a 5 N NaOH (Sigma) solution were combined. The mixture was diluted to a final volume of 50 mL for a concentration of 1 M borate. The final pH of this buffer was 9.5. Dynabeads MyOne Tosylactivated (Invitrogen, Carlsbad, CA) were resuspended and 333 μL were transferred to a clean tube. Using a magnet, the supernatant was collected then discarded. Beads were rinsed with 0.1 M borate buffer pH 9.5. Using a magnet, the supernatant was collected then discarded. Then, 400 μg of purified Clone 42 anti-alpha-synuclein antibody (BD Biosciences, San Jose, CA) was added to the beads along with 100 μL of 1 M borate buffer pH 9.5. The mixture was diluted to a final volume of 1 mL. The beads were incubated by rotating overnight at room temperature. After the overnight incubation, 50 μL of 1 M Tris-HCl pH 8.0 (Invitrogen) were added for an additional one hour at room temperature with gentle rotation. Using a magnet, the supernatant was collected then discarded, and the beads were washed with 1 mL phosphate-buffered saline (PBS, Invitrogen) buffer three times. Beads were resuspended in 1 mL PBS and stored at 4°C.
8
Magnetic Bead-based Mycobacteriophage Capture
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Mycobacteriophage D29 was used as ligand for coating magnetic beads as previously descried by Foddai and Grant30 (link) in their recent publication. This attachment was fortified by covalent bonds between free amines on the surface of bacteriophage and tosyl groups on Tosylactivated magnetic beads. Briefly, 10 mg Dynabeads MyOne Tosylactivated (Thermo Fisher, code 65501, Milan, Italy) were transferred into Eppendorf (2 mL), washed two times with 1 mL sodium carbonate decahydrate buffer (VWR, Milan, Italy) at concentration of 0.1 M and pH = 9.5 as binding buffer, and magnetically recovered. Then, beads were resuspended at the same binding buffer, exposed to mycobacteriophage D29 (Actiphage, UK, England) at concentration of 108 pfu/mL, and incubated at 37 °C overnight rotating (10–20 rpm) continually. A day after, beads were recovered magnetically, washed two times with the binding buffer, and resuspended in Middlebrook 7H9 medium supplemented with 10% OADC and 2 mM CaCl2. At the end, the phage-bead suspension was stored at 4 °C and only fifteen minutes prior inoculation transferred into the room temperature in order to be equilibrated with this temperature.
9
Peptide-Mediated Magnetic Separation of MAP
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In order to select, concentrate and separate MAP from other non-target bacteria and inhibitors possibly present in milk, subsamples of this matrix were subjected to peptide-mediated magnetic separation (PMS) from aliquots obtained before and after the copper ion challenge. Briefly, this technique uses paramagnetic beads (Dynabeads® MyOne™ Tosylactivated, Thermo Fisher) coated with two biotinylated peptides (aMp3 and aMptD) with specific binding to MAP, maximizing its capture and avoiding the non-specific recovery of other mycobacteria. Peptide-mediated magnetic separation was performed on 1 mL aliquots of milk in a BeadRetriever™ (Invitrogen, Waltham, MA, USA) according to the protocol published [22 (link)].
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