293FT cells were transfected with HA-tagged NTCP and Myc-tagged KIF4 expression plasmids at a 1:1 ratio for the assessment of the possible physical interaction between NTCP and KIF4. At 72 h after transfection, the cells were lysed and subjected to immunoprecipitation with the mouse monoclonal anti-Myc (Santa Cruz) antibody or mouse normal IgG as a negative control. Following IP, the samples were subjected to immunoblotting to detect co-IP HA-NTCP and α-tubulin (microtubule marker) in the pull-down fraction. Cell lysis and co-IP were conducted using the Pierce co-IP Kit (Thermo Fisher Scientific, 26149) according to the manufacturer’s instructions. Recombinant NTCP and KIF4 proteins were co-incubated in PBS at 4°C for 8 hours; co-IP were conducted using the Pierce co-IP Kit (Thermo Fisher Scientific, 26149) according to the manufacturer’s instructions.
Pierce co ip kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Co-IP kit is a laboratory product designed for the co-immunoprecipitation (co-IP) technique. The kit provides the necessary reagents and components to perform co-IP experiments, which are used to identify protein-protein interactions within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (4 mentions)
Anti flag antibody, by Thermo Fisher Scientific (3 mentions)
Anti hdgf, by Proteintech (3 mentions)
Aminolink plus coupling resin, by Santa Cruz Biotechnology (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Non denaturing lysis buffer, by Thermo Fisher Scientific (3 mentions)
Anti c jun, by Proteintech (2 mentions)
Genesys 10 uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Immunocruz western blotting luminol reagent, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti ha antibody, by Thermo Fisher Scientific (1 mentions)
Anti nap1l1, by Abcam (1 mentions)
Rabbit monoclonal anti met antibody, by Cell Signaling Technology (1 mentions)
Anti egfr antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti akt antibody, by Cell Signaling Technology (1 mentions)
Protein a g bead, by Thermo Fisher Scientific (1 mentions)
122 protocols using pierce co ip kit
1
NTCP-KIF4 Interaction Evaluation
2
Co-immunoprecipitation of Cbl and Ubiquitin
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Co-IP was performed using Pierce Co-IP kit (Invitrogen) as instructed in the manual. MRMECs were infected with Ad-Cbl and Ad-Ubiquitin-FLAG for 24 h, then treated with 10 μM MG-132 for 6 h and lysed in RIPA lysis buffer containing 1X protease/phosphatase inhibitor cocktail and 1 mM PMSF. Anti-FLAG antibody (Invitrogen) was immobilized with AminoLink Plus coupling resin for 2 h at room temperature. Cell lysates were incubated with anti-FLAG-conjugated agarose resin at 4°C overnight. After washing, the eluted Co-IP samples were analyzed by Western blotting.
3
Co-immunoprecipitation of Cbl and Ubiquitin
4
Co-immunoprecipitation of FLAG-tagged Proteins
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Co-IP was performed using a Pierce Co-IP kit (Invitrogen) according to the manufacturer's instructions. After exposure to 300 μM H2O2, HUVECs were incubated with MG132 (10 μM) for 2 h and then lysed in IP lysis buffer containing a protease/phosphatase inhibitor cocktail and 1 mM PMSF. The anti-FLAG antibody (Thermo Fisher Scientific, 91878) was immobilized with AminoLink Plus coupling resin for 2 h at RT. Cell lysates were incubated overnight with anti-FLAG-conjugated agarose resin at 4 °C. After washing, the eluted immunoprecipitant samples were analyzed via Western blot.
5
Endogenous STAT3 Co-Immunoprecipitation
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Co-immunoprecipitation (Co-IP) was performed using a Pierce Co-IP kit (Invitrogen) according to the manufacturer's instructions, as described previously.18 (link) For immunoprecipitation of endogenous STAT3, anti-HA antibody (Invitrogen) was added to AminoLink Plus coupling resin or negative control resin. To assess ubiquitination, the protein samples were immunoprecipitated with an anti-FLAG antibody.
6
Co-Immunoprecipitation for Protein Interactions
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Co-Immunoprecipitation (Co-IP) assay was carried out using Pierce Co-IP Kit (Thermo Scientific, United States) following the manufacturer’s instructions. The total protein was extracted and quantified. A total of 3,000 μg protein in 400 μl supernatant was incubated with 10 μg anti-NAP1L1 (Abcam), anti-HDGF (Proteintech), anti-c-Jun (Proteintech), or anti-IgG antibodies for 12 h at 4°C. The beads were washed, eluted in a sample buffer, and boiled for 10 min at 100°C. The immune complexes were subjected to Western blot analysis. Anti-IgG was used as an NC. The Cat numbers, origins, and dilution concentrations used for all antibodies are listed in Supplementary Table 2 .
7
Protein Co-Immunoprecipitation Assay
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Cells were washed once with modified Dulbecco’s phosphate-buffered saline and lysed in buffer containing 0.025 M Tris, 0.15 M NaCl, 0.0001 M EDTA, 1% NP-40, and 5% glycerol (pH 7.4). Protein G/A agarose resin (Thermo Fisher Scientific, Waltham, MA) was incubated for 2 hours with rabbit monoclonal anti-MET antibody (Cell Signaling Technologies), anti-EGFR antibody (Cell Signaling Technologies) or rabbit IgG control (Invitrogen, Carlsbad, CA) as a negative control and cell lysate at 1 mg of protein was mixed with antibody-coupled resin (Cell Signaling Technologies) overnight at 4°C. Co-immunoprecipitation was performed using the Pierce Co-IP Kit (Thermo Fisher Scientific). Immunoprecipitates were washed, eluted, and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Western blot was performed as described above.
8
Co-Immunoprecipitation of AKT and SRC-3
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Co-IPs were performed according to a standard protocol using a Pierce co-IP Kit (ThermoFisher Scientific). Briefly, differently pre-treated cells were harvested in ice-cold IP Lysis/Wash Buffer, before centrifugation at 13 000 × g for 10 min to pellet the cell debris. Then, the IP was performed by the addition of antibodies to cell lysates: rabbit anti-AKT antibody (Cell Signaling Technology) to IP the SRC-3 protein, and rabbit anti-SRC-3 antibody (Cell Signaling Technology) to IP the AKT protein. All co-IP steps were performed at 4 °C unless otherwise indicated. Subsequently, protein A/G beads (Thermo Fisher Scientific) were added for an additional 2 h. The immunoprecipitated proteins were washed five times with IP Lysis/Wash Buffer. Finally, proteins were resolved by SDS/PAGE and immunoblotted with antibodies as indicated.
9
Co-IP Experiments with Self-Developed Antibodies
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Coimmunoprecipitation (Co-IP) experiments were performed using a Pierce Co-IP Kit (Thermo Fisher Scientific). The following antibodies were developed in our laboratory and used for these experiments: 5A12 and 6H8.
10
GADD45B Protein Interaction Study
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Lysate of cells treated with SFE (20 µM) or overexpressing of GADD45B was generated under the addition of Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Protein concentration was measured by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 2 550 µg/mL protein was used for co-IP assay performed with the Pierce Co-IP Kit (Thermo Fisher Scientific). 50 µg of the GADD45B primary antibody was incubated with the delivered resin and covalently coupled for 2 h. The antibody-coupled resin was incubated with 250 µL cell lysates overnight at 4 °C, and then the protein complexes were eluted. Subsequent western blotting assay was performed as described before.
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