For coimmunoprecipitation, the cells were resuspended in lysis buffer (50mM HEPES pH7.5, 150mM NaCl, 2mM EDTA, 0.5% NP-40), supplemented with protease inhibitors (Halt Protease Inhibitor Mix, Thermo). An equal volume of acid-washed glass beads was added to the mixture, and samples were vortexed 5 for 2 min, then kept on ice for 5 mins in between. The crude extract was collected and cleared by centrifugation at maximum speed twice for 15 minutes at 4°C. An equal volume of the cleared lysate was mixed with Protein A or G-agarose beads depending on the primary antibody (Roche) pre-incubated with the antibody of interest. After the overnight incubation, the beads were washed twice with 1ml lysis buffer containing protease inhibitors, and the samples were eluted in 100 μl 6X Laemmli buffer containing 0.9% B-mercaptoethanol by boiling at 95°C for 5 minutes. Then, the samples were centrifuged to precipitate the beads, and the soluble part was used to run SDS-PAGE gels.
Halt protease inhibitor mix
Manufactured by Thermo Fisher Scientific
Halt Protease Inhibitor Mix is a laboratory reagent designed to inhibit a wide range of proteases, including serine, cysteine, and metalloproteases. It is intended for use in the preparation and stabilization of protein samples prior to analysis.
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Lab products found in correlation
4 protocols using halt protease inhibitor mix
1
Coimmunoprecipitation Assay Protocol
2
Protein-Protein Interaction Analysis by Nanoluciferase Assay
3
Co-immunoprecipitation of HA-BCA3 Complexes
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Co-immunoprecipitation was carried out as previously described [12 (link),17 (link)]. Briefly, HEK-293 cells were grown on 100 mm plates and transfected with HA-BCA3 or HA-Δ6BCA3. At 48 h post-transfection, the cells were washed with PBS and lysed in 500 µL CO-IP buffer B (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM MgCl2, 0.5% NP-40) containing Halt Protease Inhibitor Mix (Thermo Scientific) for 30 min on ice. One-fifth of the total cell lysate sample was used for Western blot analysis. The rest of the cell lysate was cleared by centrifugation and diluted with 1 mL CO-IP A buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM MgCl2). The primary antibody, monoclonal anti-PKA(C) or anti-HA, was added to the cell extract and incubated overnight at 4 °C. The next day, 20 µL Protein-A/G-Sepharose beads were added, and after 2 h incubation at 4 °C, the immunocomplexes were collected by centrifugation. The pellets were washed with CO-IP A buffer and PBS, and immunoprecipitated proteins were separated by SDS–PAGE, transferred onto a nitrocellulose membrane, and detected by Western blot analysis.
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Co-immunoprecipitation of Viral Proteases
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Co-immunoprecipitation was carried out as previously described [54 (link)]. Briefly, HEK-293 or HeLa cells were grown on 60 mm plates and co-transfected with HA-BCA3, c-myc-M-PMV PR17(D26N), c-myc M-PMV PR12(D26N) or c-myc-HIV-1 PR(D25N). After 48 h, the cells were washed 1× with PBS and lysed in 300 μl CO-IP buffer B (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM MgCl2, 0.5% NP-40) containing Halt Protease Inhibitor Mix (Thermo Scientific) for 30 min on ice. A 1/5 of the total cell lysate sample was used for Western blot analysis. The rest of the cell lysate was cleared by centrifugation and diluted with 1 200 μl CO-IP A buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM MgCl2). Primary antibody [monoclonal anti-c-myc (Santa-Cruz), rabbit anti-c-myc or EZview Red Anti-HA Affinity Gel (Sigma)] was added to the cell extract and incubated overnight at 4°C. Optionally, 20 μl Protein-A/G-Sepharose beads were added, and after 2 h incubation at 4°C, the immunoprecipitates were collected by centrifugation. The pellets were washed 3× with CO-IP buffer and then 1× with PBS. Proteins were resolved by SDS-PAGE and analyzed by Western blotting.
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