Cells were lysed in 500 μl co-immunoprecipitation assay (co-IP) lysis buffer (Thermo Scientific) on ice for 30 min. The protein extracts were incubated with anti-IgG, -Smad2, -Smad3, or -Smad4 antibodies at 4°C for 2 h, followed by the addition of 10 μl protein A/G-agarose beads (Santa Cruz Biotechnology) and incubation at 4°C overnight. Immunoprecipitates were washed three times with RIPA buffer and then resuspended and boiled in SDS loading buffer. Western blotting was performed using an anti-RGC-32 antibody generated by us [19 (link)].
Co ip lysis buffer
Manufactured by Thermo Fisher Scientific
Co-IP lysis buffer is a solution designed for cell lysis and extraction of protein complexes for co-immunoprecipitation (Co-IP) experiments. It provides a gentle and effective means of disrupting cell membranes and releasing protein complexes while maintaining their interactions.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Protein a g agarose beads, by Cytiva (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Turbofect, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti phd2, by Novus Biologicals (1 mentions)
Mg132, by Santa Cruz Biotechnology (1 mentions)
Anti hif1a, by Abcam (1 mentions)
3 protocols using co ip lysis buffer
1
Co-immunoprecipitation of Smad proteins
2
Butein Modulates MDM2-p53 Interaction and Ubiquitination
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For MDM2–p53 interaction experiments, after treatment with butein the HepG2 cells in 100 mm dish were lysed with CO-IP lysis buffer (#87787; Thermo Scientific) containing protease inhibitors. 2 μg p53 antibodies and 100 μL protein A/G agarose beads (Amersham Biosciences AB, Buckinghamshire, UK) were added into the lysates and incubated at 4°C overnight, and then the beads were collected by centrifugation. The precipitated proteins on the beads were washed at least three times and mixed with the loading buffer, boiled at 100°C for 5 min, subjected to SDS-PAGE and then probed with anti-MDM2 antibody. For p53 ubiquitination analysis, the HepG2 cells in the 100 mm dish were treated with butein and lysed with RIPA lysis buffer (#89900; Thermo Scientific) containing protease inhibitors; appropriate p53 antibodies and 100 μL protein A/G agarose beads (Amersham Biosciences AB) were added into the lysates and incubated at 4°C overnight, then the beads were collected by centrifugation. The precipitated proteins on the beads were washed at least three times and mixed with the loading buffer, boiled at 100°C for 5 min, and subjected to SDS-PAGE, and p53 ubiquitination was detected with an anti-ubiquitin antibody.
3
HIF1α Regulation by RASSF1A
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HEK293 cells were grown to 80% confluence in 100 mm dishes and then, transfected with various plasmids (RASSF1A-FLAG, RASSF1A-FLAG (S203A), RASSF1A-FLAG (S203D) or HIF1A) using Turbofect (Thermo Scientific). 6 h after transfection, cells were subjected to 24 h hypoxia or normoxia, followed by cell lysate preparation in Co-IP lysis buffer (Thermo Scientific). Addtionally, for imunoprecipitations carried out to check post-translational modifications (prolyl-hydrxylation, lys48 ubiquitination) of HIF1α, cells were pretreated with MG132 (Santa Cruz) for 30 min before hypoxia exposure, followed by 6 h hypoxia exposure and lysis in Co-IP lysis buffer. Lysates were centrifuged and supernatants were further used for CoIP. Equal amounts of proteins were incubated overnight on rotation with anti FLAG (Sigma), anti-HIF1A (abcam), anti-PHD2 (Novus biological) or IgG (Santa Cruz), followed by 3 h incubation with Protein G sepharose beads (GE healthcare). After incubation, beads were repeatedly washed with PBS + 0.1% tween20, followed by addition of SDS sample buffer and analysis by western blotting.
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