Dynabeads m 280 sheep anti rabbit igg magnetic beads

Primary antibodies against FOXC1, EZH2, SUZ12 and IgG isotype control were incubated overnight with Dynabeads M-280 Sheep anti-Rabbit IgG magnetic beads (Invitrogen) followed with washing using Washing Buffer (50 mM Tris-HCl pH 7.5, 137 mM NaCl, 0.005 Triton X-100, 1 mM EDTA, de-ionized H₂O). Whole cell lysates from cultured cells were incubated with the primary antibody-bound magnetic beads under continuous rotation at 4°C for 4 h. Excessive unbound protein was cleared from the beads by washing. After 5 rounds of washing with Washing Buffer, the co-IP products were eluted from the beads by boiling in 1x SDS Loading Buffer at 99°C for 5 min with shaking at 1000 rpm. Three percent of the whole cell lysate input was loaded alongside co-IP products for gel electrophoresis. Nitrocellulose membrane bound with protein was incubated with the corresponding primary antibodies followed by incubation with the sheep-against rabbit light-chain specific secondary antibody. Developed membrane was imaged using ChemiDoc (Bio-Rad) as described above.