Protein a g agarose beads slurry

Pull-down assay was performed to test direct binding between purified recombinant proteins. 5 μL of glutathione agarose (GST pull-down; Pierce), HisPur Ni-NTA resin (His pull-down; Pierce), or 15 ul of Protein-A/G agarose beads slurry (SantaCruz) cross-linked with CatSper1 antibody was equilibrated with pre-binding buffer (10 mM HEPES pH7.5, 140 mM NaCl), and incubated with the recombinant GST-tagged EFCAB9 or 6xHis tagged CatSperζ, or GST-CatSper1-N150 protein (bait proteins), respectively, at 4°C for overnight. Incubated resin was washed in pre-binding buffer three times and equilibrated in the binding buffer in various compositions for each experiment. Prey proteins subjected to interact with bait proteins were incubated with the resin equilibrated with binding buffer for 1 hr at RT. The resin was washed in the binding buffer for each group four times. Resin was collected and the bound proteins were eluted with 2X LDS sampling buffer supplemented with 50 mM DTT, and denatured at 75°C for 10 minutes. Protein interaction was confirmed by Coomassie blue staining (GelCode^Tm Blue Safe Protein Satin, ThermoFisher) or western blotting. Detailed experiment procedures and compositions of binding buffers for each experiment are as described below.