Immunoprecipitation was conducted with protein G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Cells after transfection were lysed and incubated in immunoprecipitation lysis buffer (Beyotime) for 10 min at room temperature. The extracts were incubated with Notch1-antibody (2 μg/ml, Abcam, Cambridge, UK) at 4 °C overnight, and the immunoprecipitate was purified by protein G agarose beads with gentle rocking. The beads were washed for three times with extraction buffer and resuspended in 20 μl SDS loading buffer. The whole cell lysates and immunoprecipitates were incubated at 70 °C for 10 min followed with western blot analysis.
Notch1 antibody
Manufactured by Abcam
Sourced in United Kingdom
Notch1-antibody is a laboratory reagent used for the detection and analysis of the Notch1 protein in biological samples. Notch1 is a transmembrane receptor that plays a key role in cellular signaling pathways. The Notch1-antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to study the expression and localization of Notch1 in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Immunoprecipitation lysis buffer, by Beyotime (1 mentions)
Protein g agarose beads, by Abcam (1 mentions)
Protein g agarose bead, by Thermo Fisher Scientific (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Recombinant mouse dll4, by BioLegend (1 mentions)
Rbpj antibody, by Cell Signaling Technology (1 mentions)
2 protocols using notch1 antibody
1
Notch1 Immunoprecipitation Protocol
2
Investigating NOTCH1 and RBPJ Binding in EL4 Cells
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EL4 cells were treated with PBS or 5 µg/ml recombinant mouse DLL4 (BioLegend, #776706) for 24 hours before crosslinked with 1% formaldehyde for 10 minutes at room temperature.
Reaction was quenched with 125 mM glycine. ChIP was performed as previously described (Zhao et al., 2018) with NOTCH1 antibody (Abcam, ab27526), RBPJ antibody (Cell Signaling Technology, #5313), or normal rabbit IgG (Cell Signaling Technology, #2729). The sequences of all ChIP primers are listed in Supplemental Table 2.
Reaction was quenched with 125 mM glycine. ChIP was performed as previously described (Zhao et al., 2018) with NOTCH1 antibody (Abcam, ab27526), RBPJ antibody (Cell Signaling Technology, #5313), or normal rabbit IgG (Cell Signaling Technology, #2729). The sequences of all ChIP primers are listed in Supplemental Table 2.
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