GATA6Ex4Δ1/Δ2:ind GATA6 cells were differentiated to mesendoderm (day 2). Between days 1 and 2, the cells were cultured in the absence or presence of 10ng/ml doxycycline to induce GATA6–3xFLAG expression at endogenous levels. At Day 2, cells were washed once with PBS and lysed with IP lysis buffer (ThermoFisher/Pierce, IL, #87787) with HALT proteinase inhibitor cocktail (ThermoFisher/Pierce, IL, #78443) and Universal Nuclease (ThermoFisher/Pierce, IL, #88701). The sample was incubated at room temperature for 10 minutes before centrifugation to remove cell debris. The lysate was pre-cleared with pre-washed Protein G Dynabeads (ThermoFisher Scientific, NY, #10004D) for 2 hours at 4°C. An input sample was taken and the remaining lysate split equally and incubated with either 1 μg of FLAG M2 (Sigma-Aldrich, MO, #F1804) or 1 μg whole mouse IgG (Sigma-Aldrich, MO, #12–371) antibodies overnight at 4°C. The following day, pre-washed Protein G Dynabeads were added and the mixture was incubated for 2 hours at 4°C. The beads were then washed 5 times with IP lysis buffer and eluted in 1x Lamelli buffer (BioRad, CA, #1610747) at 95°C for 10 minutes. Samples were then analyzed by western blot. Antibodies used for co-IP listed in Table S8 .
Ip lysis buffer
Manufactured by Bio-Rad
Sourced in United States
IP lysis buffer is a solution used to lyse cells and extract proteins for immunoprecipitation (IP) experiments. The buffer contains a combination of detergents, salts, and buffering agents to effectively disrupt cell membranes and solubilize cellular proteins while maintaining protein-protein interactions. This buffer is designed to preserve the native conformation and interactions of the target proteins during the IP process.
Automatically generated - may contain errors
Lab products found in correlation
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Whole mouse igg, by Merck Group (2 mentions)
Lamelli buffer, by Bio-Rad (2 mentions)
Flag m2, by Merck Group (2 mentions)
Anti sirt1 antibody, by Cell Signaling Technology (1 mentions)
Protein a g magnetic beads, by Selleck Chemicals (1 mentions)
Laemmli protein sample buffer, by Bio-Rad (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti vimentin antibody, by Proteintech (1 mentions)
3 protocols using ip lysis buffer
1
GATA6 Protein Co-Immunoprecipitation
2
Immunoprecipitation of SIRT1 and Vimentin
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Anti‐SIRT1 antibody (Cell Signaling Technology, #8469), anti‐vimentin antibody (Proteintech, #10366‐1), or matched IgG isotype antibody (Cell Signaling Technology, #5946) were incubated with Protein A/G Magnetic Beads (Bimake, Houston, USA, #B23202) for 1.5 h at room temperature. Cells and tissues were lysed with IP lysis buffer (Thermo Fisher Scientific, Waltham, USA, #87787), and a total of 400 μg of each sample lysate was incubated with an antibody−bead complex for another 8 h. Then, the protein extracts were precipitated by the antibody−bead complex using a magnetic rack. After being washed 3 times with IP lysis buffer, Co‐IP products were boiled at 95°C for 15 min with diluted 4 × Laemmli protein sample buffer (Bio‐Rad, Hercules, USA, #1610747) in lysis buffer. Finally, proteins were resolved by SDS/PAGE and immunoblotted with antibodies as indicated.
3
GATA6 Protein Co-Immunoprecipitation
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GATA6Ex4Δ1/Δ2:ind GATA6 cells were differentiated to mesendoderm (day 2). Between days 1 and 2, the cells were cultured in the absence or presence of 10ng/ml doxycycline to induce GATA6–3xFLAG expression at endogenous levels. At Day 2, cells were washed once with PBS and lysed with IP lysis buffer (ThermoFisher/Pierce, IL, #87787) with HALT proteinase inhibitor cocktail (ThermoFisher/Pierce, IL, #78443) and Universal Nuclease (ThermoFisher/Pierce, IL, #88701). The sample was incubated at room temperature for 10 minutes before centrifugation to remove cell debris. The lysate was pre-cleared with pre-washed Protein G Dynabeads (ThermoFisher Scientific, NY, #10004D) for 2 hours at 4°C. An input sample was taken and the remaining lysate split equally and incubated with either 1 μg of FLAG M2 (Sigma-Aldrich, MO, #F1804) or 1 μg whole mouse IgG (Sigma-Aldrich, MO, #12–371) antibodies overnight at 4°C. The following day, pre-washed Protein G Dynabeads were added and the mixture was incubated for 2 hours at 4°C. The beads were then washed 5 times with IP lysis buffer and eluted in 1x Lamelli buffer (BioRad, CA, #1610747) at 95°C for 10 minutes. Samples were then analyzed by western blot. Antibodies used for co-IP listed in Table S8 .
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