Journal: bioRxiv
Article Title: Uncovering protein-protein interactions of the human sodium channel Nav1.7
doi: 10.1101/2023.04.18.537407
Figure Lengend Snippet: A . Schematic illustrating the procedures involved in the affinity purification, SEC, and MS/MS of TAP-tagged hNav1.7. B . TAP-tagged NaV1.7 was detected using western blotting with anti-FLAG antibody after single-step and tandem affinity purification (SS-AP and TAP). Ci . A representative size exclusion chromatography of the protein sample. Cii . The resolved fractions isolated from size exclusion chromatography visualized using Coomassie blue staining. D . The validation of selected Nav1.7 protein interactor candidates Kif11, CCT5, and TMED10, where Nav1.7 complexes were immunoprecipitated with anti-FLAG M2 magnetic beads and then detected with their specific antibodies using Western blotting.
Article Snippet: The membranes were blocked with 5% nonfat milk for 30 minutes before being treated with primary antibodies, including anti-FLAG (1:1000, Sigma, F1804), anti-Nav1.7 (1:500, Origene, TA329033), anti-CCT5 (1:1000, Origene, TA308298), and anti-TMED10 (1:1000, Origene, TA306375), overnight at 4°C.
Techniques: Affinity Purification, Tandem Mass Spectroscopy, Western Blot, Size-exclusion Chromatography, Isolation, Staining, Immunoprecipitation, Magnetic Beads