IP for Anti-Netrin-1 [IPI-NTN1.45]

Antibody-bead conjugates were prepared by adding 2 µg of antibody to 500 µL of Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol; Thermo Fisher Scientific #87788) in a microcentrifuge tube, together with 30 µL of Dynabeads protein A (Thermo Fisher Scientific #10002D). Tubes were rocked for ~1 h at 4 °C followed by two washes to remove unbound antibodies. Culture media from WT MCF7 were collected and total protein quantified. Aliquots of 0.15 mL of culture medium, containing 1 mg/mL total protein, were incubated with the antibody-bead conjugate for ~1 h at 4 °C. The unbound fractions were collected, and beads were subsequently washed three times with 1 mL of IP buffer and processed for SDS-PAGE and western blot on precast midi 4–20% Tris-Glycine polyacrylamide gels. Protein A:HRP was used as a secondary detection system at a concentration of 0.5 µg/mL.

References

Ayoubi et al. (2025). A guide to selecting high-performing antibodies for Netrin-1 (UniProt ID: O95631) for use in western blot and immunoprecipitation. Cell. 96(6):795-806. https://doi.org/10.5281/zenodo.15596280 (Link opens in a new window)