The RNA was extracted from lymphocytes in MagNA Pure LC2.0 Automatic extractor with MagNA Pure LC Total Nucleic Acid Isolation Kit–High Performance: automatic RNA extraction using magnetic beads (Roche Diagnostics, IN, USA). The isolation protocol was performed according to the manufacturer's instructions. After extraction, RNA was quantified with a NanoDrop2000 (Thermo fisher Scientific, USA); Purity ranging between 1.8 and 2.1 was deemed as a good RNA quality.
Magnetic beads
Manufactured by Roche
Sourced in United States
Magnetic beads are small, spherical particles that can be coated with various surface functional groups. They are designed to be used in a range of laboratory applications, enabling efficient separation, purification, and manipulation of target molecules or cells.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop tablet, by Roche (2 mentions)
1 μm magnetic beads, by Polysciences (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Magna pure lc 2, by Roche (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Magna pure lc total nucleic acid isolation kit, by Roche (1 mentions)
4 protocols using magnetic beads
1
Automated Lymphocyte RNA Extraction
2
Phagosome Isolation and Protein Complex Purification
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Cells in a confluent 15cm dish were incubated with ∼108 1 μm Magnetic beads (Polysciences) for 4 h. After rigorous washing in PBS, cells were scraped into 10 ml sucrose homogenization buffer (SHB: 250 μM sucrose, 3 mM imidazole, pH 7.4) and pelleted by centrifugation. Cells were resuspended in 2 ml SHB plus protease inhibitor cocktail with EDTA (Roche) and 1mM PMSF and disrupted by 25 strokes in a steel dounce homogenizer. The disrupted cells were gently rocked for 10 min on ice to free endosomes. Beads were collected with a magnet (Dynal) and washed 4x with SHB plus protease inhibitor. After the final wash, phagosome preparations were denatured in 2x SDS buffer at room temperature for 1 h and analyzed by western blot.
For protein complex purification, phagosome preparations were lysed in NP-40 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, supplemented with 1 mM PMSF, complete protease inhibitor cocktail and PhosSTOP tablets (Roche) on ice for 1 h. Magnetic beads were removed by magnet and insoluble components were precipitated by 15,000 g spin for 20 min. Lysate was incubated with anti-FLAG matrix for 3 h, followed by four washes in lysis buffer. Proteins were eluted in NP-40 buffer containing 200 ng/ml 3xFLAG peptide, and were further applied to western blot, silver stain or Trypsin in-solution digest for mass spectrometry.
For protein complex purification, phagosome preparations were lysed in NP-40 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, supplemented with 1 mM PMSF, complete protease inhibitor cocktail and PhosSTOP tablets (Roche) on ice for 1 h. Magnetic beads were removed by magnet and insoluble components were precipitated by 15,000 g spin for 20 min. Lysate was incubated with anti-FLAG matrix for 3 h, followed by four washes in lysis buffer. Proteins were eluted in NP-40 buffer containing 200 ng/ml 3xFLAG peptide, and were further applied to western blot, silver stain or Trypsin in-solution digest for mass spectrometry.
3
Protein-DNA Interaction Assay
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Magnetic beads (Roche) bound to a 5′-biotinylated ssDNA polydT 83-mer (corresponding to the final concentration of 1 mM nucleotide) were pre-incubated with a blocking buffer (25 mM Tris–acetate pH 7.5, 40 mM KOAc, 1 mM DTT, 0.02% Igepal and 10 mg/ml BSA) for 20 min at 37°C. The beads were captured with the magnetic particle separator, washed twice with reaction buffer (25 mM Tris–Acetate pH 7.5, 40 mM KOAc, 1 mM DTT, 0.02% Igepal, 1 mg/ml BSA, 5 mM ATP and 0.5 mM MgCl2) and resuspended in the same buffer. hRPA was added and the reaction was incubated for 5 min at 37°C. Then RAD51 was added and the reaction was incubated for 5 min at 37°C. Next, APRIN was added to the reaction for a final volume of 20 μl for 30 min at 37°C. The beads were captured and were washed twice with 500 μl of washing buffer (25 mM Tris–Acetate pH 7.5, 40 mM KOAc, 1 mM DTT, 0.02% Igepal, 5 mM ATP and 0.5 mM MgCl2). Finally, 15 μl of Laemmli buffer was added and beads were heated 5 min at 95°C. The beads were captured again and the supernatants were loaded onto a 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis Ruby (Invitrogen) and visualized by ultraviolet (UV).
4
Phagosome Isolation and Protein Complex Purification
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