The IP of MIC3 from ESAs was performed as described previously with some modification59 (link). For a single IP, 20 μl mouse monoclonal antibody to T. gondii MIC3 (anti-MIC3 mAb, GeneTex) was incubated with 200 μl protein G-agarose suspension (Sigma-Aldrich, St. Louis, MO, USA) at 4 °C for 3 h. Next, anti-MIC3 mAb conjugated to the protein G-agarose suspension was collected by centrifugation, mixed with ESAs (200 μg) and incubated with mixing at 4 °C for 3 h. Finally, the protein G-agarose was removed by centrifugation. The depletion of MIC3 from ESA was analysed by ELISA. Protein concentrations were assayed using the Bradford method. Equal amounts of ESAs and MIC3-depleted ESAs were loaded.
Protein g agarose suspension
Manufactured by Merck Group
Sourced in United States
Protein G-agarose suspension is a laboratory reagent used for the purification and isolation of immunoglobulins (antibodies) from biological samples. It consists of protein G, a bacterial surface protein, covalently coupled to an agarose matrix. Protein G has a high affinity for the Fc region of various immunoglobulin classes, allowing for the specific capture and separation of antibodies from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Ip lysis buffer, by Beyotime (4 mentions)
Flag m2 affinity gel, by Merck Group (2 mentions)
Flag peptide, by Merck Group (2 mentions)
Odyssey sa infrared imaging system, by LI COR (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
P0013, by Beyotime (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Easyblot anti mouse, by GeneTex (1 mentions)
Easyblot anti rabbit, by GeneTex (1 mentions)
6 protocols using protein g agarose suspension
1
Immunoprecipitation of T. gondii MIC3
2
Protein Complex Isolation and Identification
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Cells were harvested and lysed with an IP lysis buffer (Beyotime Institute of Biotechnology, P0013). Total protein (up to 5 mg) was incubated with Flag M2 Affinity Gel (Sigma, A2220) to immunoprecipitated the Flag-tagged proteins, overnight at 4 °C on a rocking platform. The immunoprecipitates were collected by centrifugation and washed three times with the cold TBS. the immunoprecipited protein complexes were eluted through 3 × Flag peptides (Sigma, F4799). The co-immunoprecipitated proteins were detected through western blot assay or subjected to the LC-MS/MS peptide identification.
Co-immunoprecipiation of the endogenous proteins was achieved by Protein G–agarose suspension (Millipore, 16–266). Total protein was incubated with 50 μl of Protein G–agarose suspension for 3 h at 4 °C on a rocking platform to reduce non-specific binding. After removing the beads, the supernatant was supplemented with the primary antibodies followed by incubation for an additional 3 h at 4 °C. A total of 100 μl of Protein G–agarose was then added to each immunoprecipitation mixture, and the incubation was continued overnight at 4 °C on a rocking platform. The immunoprecipitates were collected by centrifugation and washed three times with the TBS. The agarose was boiled with loading buffer and subjected to western blot analysis.
Co-immunoprecipiation of the endogenous proteins was achieved by Protein G–agarose suspension (Millipore, 16–266). Total protein was incubated with 50 μl of Protein G–agarose suspension for 3 h at 4 °C on a rocking platform to reduce non-specific binding. After removing the beads, the supernatant was supplemented with the primary antibodies followed by incubation for an additional 3 h at 4 °C. A total of 100 μl of Protein G–agarose was then added to each immunoprecipitation mixture, and the incubation was continued overnight at 4 °C on a rocking platform. The immunoprecipitates were collected by centrifugation and washed three times with the TBS. The agarose was boiled with loading buffer and subjected to western blot analysis.
3
IP-Ubiquitination and Co-Immunoprecipitation Assays
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For IP-ubiquitination assay, cells were lysed with RIPA buffer (P0013K, Beyotime Biotechnology) containing 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, the protease inhibitor (05892970001, Roche Diagnostics), and phosphatase inhibitor cocktail (04906845001, Roche Diagnostics). For co-immunoprecipitation (co-IP), cells were lysed with Western and IP lysis buffer (P0013, Beyotime Biotechnology) containing the protease and phosphatase inhibitors. The supernatants of cell lysates were incubated with Protein G-agarose suspension (16-266, Millipore) together with specific antibodies. After being incubated overnight, the precipitates were washed three times with cold IP buffer and boiled with loading buffer for western blotting. Images were visualized by the ChemiDocTM XRS system (Bio-Rad) or Odyssey Sa Infrared Imaging System (LI-COR). The band intensity of western blotting was analysed by Odyssey Sa Imaging System Application Software (Version 1.1.7).
4
Immunoprecipitation and Co-immunoprecipitation
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Cells were collected and lysed with an IP lysis buffer (Beyotime Institute of Biotechnology, P0013). Total protein (up to 5 mg) was incubated with 50 μl of Protein G-agarose suspension (Millipore, 16–266,) for 3 h at 4 °C on a rocking platform to reduce non-specific binding. After removing the beads, the supernatant was supplemented with the primary antibodies followed by incubation for an additional 3 h at 4 °C. A total of 100 μl of Protein G-agarose was then added to each immunoprecipitation mixture, and the incubation was continued overnight at 4 °C on a rocking platform. The immunoprecipitates were collected by centrifugation and washed three times with the cold 1 × TBS. After the loading buffer was added, the agarose was boiled and subjected to western blot analysis. The EasyBlot anti-mouse (GTX221667-01) or EasyBlot anti-rabbit (GTX221666-01) IgG HRP-conjugated secondary antibodies (Genetex) were employed to avoid the denatured heavy and light chains from antibodies used in immunoprecipitation assays.
The Flag M2 Affinity Gel (Sigma, A2220) was used to immunoprecipited the Flag-tagged proteins, After three times' washes with cold 1 × TBS the immunoprecipited protein complexes were eluted through 3 × Flag peptides (Sigma, F4799). The co-immunoprecipated proteins were detected through western blot assay.
The Flag M2 Affinity Gel (Sigma, A2220) was used to immunoprecipited the Flag-tagged proteins, After three times' washes with cold 1 × TBS the immunoprecipited protein complexes were eluted through 3 × Flag peptides (Sigma, F4799). The co-immunoprecipated proteins were detected through western blot assay.
5
Immunoprecipitation Assay for Protein Interactions
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Cells were collected and lysed with an IP lysis buffer (Beyotime Institute of Biotechnology). Total protein was incubated with Protein G-agarose suspension (Millipore) for 3 hrs at 4°C on a rocking platform to reduce non-specific binding. After removing the beads, the supernatant was supplemented with the primary antibodies followed by incubation for an additional 3 hrs at 4°C. Protein G-agarose was then added to each immunoprecipitation mixture, and the incubation was continued overnight at 4°C on a rocking platform. The immunoprecipitates were collected by centrifugation and washed three times. After the loading buffer was added, the agarose was boiled and subjected to Western blot analysis.
6
Immunoprecipitation Assay in PDAC Cells
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Co-IP was performed as described previously.30 (link) In brief, PDAC cells were collected and lysed with IP lysis buffer (Beyotime, Shanghai, China). Total protein was incubated with 50 μl of Protein G-agarose suspension (Millipore, USA). After supplementation for 2 h at 4 °C, the beads were removed, and the supernatant was incubated with the primary antibodies for an additional 2 h at 4°C. Then, 100 μl of Protein G-agarose was added. After incubation at 4 °C overnight, the immunoprecipitates were collected and washed three times with 1×PBS. Finally, the loading buffer was added, and the agarose was boiled and subjected to Western blotting analysis.
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