Per experiment, 100 μL UltraLink streptavidin bead slurry (Pierce) was centrifuged, and the supernatant removed. After washing with lysis buffer (50 mM Tris–HCl, 100 mM NaCl, 0.2% NP-40, 5% glycerol, 1.5 mM MgCl2, 25 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol (DTT), 10 μg/mL TLCK, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 10 μg/mL soybean trypsin inhibitor (Sigma), pH 7.5), biotin-conjugated OICR-9429 derivative (0.05 μmol) was added and incubated on a roto-shaker for 30 min at 4 °C. After centrifugation and one additional washing step, beads were re-suspended in cell lysates (10 mg total protein per pull-down) and incubated on a roto-shaker for 2 h at 4 °C. For competition experiments, lysates were pre-incubated with 10 μM of unmodified OICR-9429, for 20 min at 4 °C. After centrifugation, beads were transferred to spin columns (MoBiTec), washed with lysis buffer and HEPES, followed by elution of bound proteins with elution buffer (50% 6 M urea, 50% 100 mM formic acid).
Spin columns
Manufactured by MoBiTec
Spin columns are a type of lab equipment used for separation and purification of biomolecules. They contain a porous medium that allows for the selective retention and elution of target analytes based on size, charge, or other properties.
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Lab products found in correlation
2 protocols using spin columns
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Affinity Purification of Protein Complexes
2
Affinity Purification of Protein Complexes
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Per experiment, 100 μL UltraLink streptavidin bead slurry (Pierce) was centrifuged, and the supernatant removed. After washing with lysis buffer (50 mM Tris–HCl, 100 mM NaCl, 0.2% NP-40, 5% glycerol, 1.5 mM MgCl2, 25 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol (DTT), 10 μg/mL TLCK, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 10 μg/mL soybean trypsin inhibitor (Sigma), pH 7.5), biotin-conjugated OICR-9429 derivative (0.05 μmol) was added and incubated on a roto-shaker for 30 min at 4 °C. After centrifugation and one additional washing step, beads were re-suspended in cell lysates (10 mg total protein per pull-down) and incubated on a roto-shaker for 2 h at 4 °C. For competition experiments, lysates were pre-incubated with 10 μM of unmodified OICR-9429, for 20 min at 4 °C. After centrifugation, beads were transferred to spin columns (MoBiTec), washed with lysis buffer and HEPES, followed by elution of bound proteins with elution buffer (50% 6 M urea, 50% 100 mM formic acid).
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