For membrane and cytosolic fractionation, we followed our previously described protocol [44 (link)]. For immunoprecipitation, 1 mg of cell lysate was incubated with 1 μg/mL of antibodies at 4 °C overnight. Immunocomplex was precipitated using protein A or G sepharose beads (Thermo Fisher Scientific). Sepharose beads were resuspended in SDS loading buffer and separated by SDS-PAGE and visualized by Western blotting. For in vitro pull-down assay, 1 μg of FC-fusion CD177 (14501-H02H, SinoBiological, Beijing, China) and His-Tag full-length β-Catenin (11279-H20B, SinoBiological), both purified from HEK293T cells, were incubated using RIPA buffer, with or without the presence of 1 mg of cell lysates from MCF-7 or MDA-MB-231 cells. Ni-NTA agarose was used to pull down His-β-Catenin complex, following with SDS-PAGE and Western Blotting.
Protein a or g sepharose beads
Manufactured by Thermo Fisher Scientific
Protein A or G Sepharose beads are bead-based resins used for the purification and isolation of antibodies or antibody-containing samples. These beads contain covalently immobilized protein A or protein G, which have a high affinity for the Fc region of immunoglobulins, allowing for the efficient capture and recovery of antibodies from complex mixtures.
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2 protocols using protein a or g sepharose beads
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Membrane Fractionation and Protein Interactions
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Membrane Fractionation and Protein Interactions
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For membrane and cytosolic fractionation, we followed our previously described protocol [44 (link)]. For immunoprecipitation, 1 mg of cell lysate was incubated with 1 μg/mL of antibodies at 4 °C overnight. Immunocomplex was precipitated using protein A or G sepharose beads (Thermo Fisher Scientific). Sepharose beads were resuspended in SDS loading buffer and separated by SDS-PAGE and visualized by Western blotting. For in vitro pull-down assay, 1 μg of FC-fusion CD177 (14501-H02H, SinoBiological, Beijing, China) and His-Tag full-length β-Catenin (11279-H20B, SinoBiological), both purified from HEK293T cells, were incubated using RIPA buffer, with or without the presence of 1 mg of cell lysates from MCF-7 or MDA-MB-231 cells. Ni-NTA agarose was used to pull down His-β-Catenin complex, following with SDS-PAGE and Western Blotting.
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