Coimmunoprecipitation (Co-IP) was performed using the Thermo Scientific Pierce Co-IP kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Ten micrograms each of antibody and IgG were immobilized on AminoLink Plus coupling resin for 2 hours, then washed and incubated with 250 μg gastric cancer cell protein lysate. After overnight incubation at 4°C, the resin was washed and eluted using an elution buffer. The eluted proteins were separated by SDS-PAGE and immunoblotted with indicated antibodies as Western blot analysis.
Thermo scientific pierce co ip kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Scientific Pierce Co-IP kit is a laboratory product designed for co-immunoprecipitation (co-IP) experiments. It provides the necessary components for the isolation and identification of protein-protein interactions.
Automatically generated - may contain errors
Lab products found in correlation
Lats1, by Cell Signaling Technology (1 mentions)
Crabp2, by Proteintech (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Anticar, by Abcam (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
Anti sirt6, by Cell Signaling Technology (1 mentions)
Anti nrf2, by Cell Signaling Technology (1 mentions)
Anti p53, by Abcam (1 mentions)
9 protocols using thermo scientific pierce co ip kit
1
Coimmunoprecipitation of Gastric Cancer Proteins
2
Coimmunoprecipitation of FOXA1, GATA3, and SOD2
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Coimmunoprecipitation (co-IP) was performed using the Thermo Scientific Pierce co-IP kit (Thermo Fisher, 26149). Briefly, FOXA1 (Abcam, ab170933) and GATA3 (Proteintech, 66400-1 Ig) antibodies were immobilized for 2 h using AminoLink Plus coupling resin. The resins were washed and incubated with NLS-SOD2 or NES-SOD2 cell lysate overnight. After incubation, proteins were eluted, quantified, and analyzed by Western blotting using anti-Flag (anti-DYKDDDK Tag) (Cell Signaling, 2368).
3
Protein Co-Immunoprecipitation Protocol
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Cells were lysed in Triton X-100 lysis buffer [NaCl (150 mM), NP-40 (0.5%), Tris-HCl (pH = 8.0), glycerol (20 mM, 20%)] containing phosphatase inhibitors and protease inhibitor (Roche, NJ, USA), then the proteins extract were centrifugated at 12,000 × g for 20 min. Thermo Scientific Pierce Co-IP kit (Thermo Fisher Scientific) was used for Co-IP experiments. At last, obtained proteins were resuspended in 5× SDS sample loading buffer, heated to 100 °C for 8 min, and tested by 10% SDS–polyacrylamide gel electrophoresis (PAGE).
4
Immunoprecipitation and Western Blot
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Proteins extract were obtained from immunoprecipitation lysis buffer [NaCl (150 mM), NP-40 (0.5%), Tris-HCl (pH = 8.0), glycerol (20 mM, 20%)] containing phosphatase inhibitors and protease inhibitor (Roche, NJ, USA) and then were centrifuged 20 min (12,000 rpm, 4 °C). Thermo Scientific Pierce Co-IP kit (Thermo Fisher Scientific) was used for Co-IP experiments. These obtained proteins were tested by SDS-PAGE (10%) and immunoblotted with Lats1 (Cell Signaling Technology) and CRABP2 (Proteintech) antibodies as Western blotting analysis.
5
Protein-Protein Interaction Analysis
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Following the manufacturer's protocol, co‐immunoprecipitation (Co‐IP) assays were conducted using the Thermo Scientific Pierce Co‐IP Kit (Thermo Scientific). The immunoprecipitated proteins were analysed by Western blot.
6
Immunoprecipitation-Based Protein Extraction and Analysis
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Proteins extract were obtained from immunoprecipitation lysis buffer (P0013, Beyotime Biotechnology) with the Complete Protease Inhibitor Cocktail, and then centrifuged for 20 min (12,000 rpm,4°C). Soluble lysates were incubated with the antibodies at 4°C overnight, followed by incubation of Protein A/G Plus-Agarose(Santa Cruz Biotechnology, TX, USA) for 2 hr. Complexes were isolated from the beads and boiled for 10 minutes. Thermo Scientific Pierce Co-IP kit (Thermo Fisher Scientific) was used for the Co-IP assay. These obtained proteins were tested by Western blotting analysis.
7
Co-Immunoprecipitation of TRIM22 and NOD2
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Cell lysates (approximately 1.5 mg total protein) were collected from the TRIM22-overexpressing (TRIM22 OE) Ishikawa cells using ice-cold non-denaturation lysis buffer. co-IP was performed according to the manufacturer's protocol of the Thermo Scientific Pierce Co-IP kit (Thermo Fisher Scientific, Inc.). TRIM22 and NOD2 antibodies were fixed for 2 h with AminoLink Plus coupling resin at room temperature. The resin was then washed and incubated with the cell lysates overnight at 4°C. The following day, the resin was again washed, and elution buffer was used to elute the protein combined with the resin. A negative control, harvested from the vector control-transfected Ishikawa cells, were treated in the same manner as the Co-IP samples, including incubation with AminoLink Plus coupling resin combined with TRIM22 and NOD2 antibodies (Novus Biologicals, LLC) overnight at 4°C. This control allowed us to observe whether the binding protein was increased in the TRIM22 OE groups. Samples were analyzed by western blot analysis using rabbit polyclonal anti-NOD2 (Novus Biologicals, LLC) and mouse monoclonal anti-TRIM22 (Novus Biologicals, LLC) antibodies.
8
Co-Immunoprecipitation of CAR and YAP in HepG2 Cells
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HepG2 cells (ATCC, Cat# HB-8065, RRID: CVCL_0027, VA, USA) were cultured in DMEM containing 10% FBS. Co-immunoprecipitation (Co-IP) was conducted using Thermo Scientific Pierce co-IP kit (Thermo Scientific) as described in our previous report24 (link). Samples were analyzed by Western blot using anti-IgG (Abcam, Cat# ab133470), anti-CAR (Abcam, Cat# ab62590, RRID: AB_956175 ) and anti-YAP (Cell Signaling Technology, Cat# 14074, RRID: AB_2650491 ).
9
Co-IP of SIRT6, P53, and NRF2
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AML12 cells were cultured according to the method described above and treated with or without APAP solution for 24 h. The protein was extracted following the protocol. Co-immunoprecipitation (Co-IP) was performed using Thermo Scientific Pierce Co-IP kit (Thermo Fisher Scientific, Rockford, IL, USA) according to our previous reported procedures29 (link). Samples were analyzed by Western blotting using anti-IgG (Merk, Kenilworth, NJ, USA, AP124), anti-SIRT6 (Cell Signaling Technology, Shanghai, China, 12486), anti-P53 (Abcam, Cambridge, MA, China, ab26) and anti-NRF2 (Cell Signaling Technology, 12721).
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