过表达细胞体系、蛋白互作专用的「材料与方法」模板，可直接复制进 SCI 论文！

格式、术语、逻辑都给你写好了，你只需要改蛋白名即可。

细胞培养与质粒共转染

Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at37 °C in a humidified incubator with 5% CO₂.For transient transfection, cells were seeded in 6-well plates or culture dishes and transfected at approximately 70–80% confluence using a commercial transfection reagent according to the manufacturer’s instructions.Cells were co-transfected with equal amounts of plasmid DNA encoding Flag-tagged Protein A and Myc-tagged Protein B, as indicated in each experiment.

Co-IP 免疫共沉淀（正反互作）

At 48 h post-transfection, cells were harvested and lysed in IP lysis buffer containing protease inhibitor cocktail on ice for 30 min.Cell lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatants were collected.For co-immunoprecipitation, lysates were incubated with anti-Flag antibody or anti-Myc antibody overnight at 4 °C with gentle rotation, followed by incubation with protein A/G agarose beads for 4 h.Beads were washed three times with lysis buffer, and bound proteins were eluted with SDS loading buffer and boiled for 5 min before Western blot analysis.

三、Western Blot 检测

Immunoprecipitated or whole-cell lysate samples were separated by SDS-PAGE and transferred to PVDF membranes.Membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated with primary antibodies against Flag, Myc, or β-actin overnight at 4 °C.After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature.Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system.

Immunofluorescence / 荧光共定位

Cells grown on coverslips were co-transfected with GFP-Protein A and mCherry-Protein B.At 48 h post-transfection, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA.Nuclei were stained with DAPI.Fluorescence images were captured using a laser scanning confocal microscope.Colocalization analysis was performed using ImageJ software.

GST/His Pull-Down（直接互作）

Recombinant GST-tagged Protein A and His-tagged Protein B were expressed and purified from Escherichia coli.For pull-down assays, GST or GST-Protein A immobilized on glutathione sepharose beads was incubated with His-Protein B for 2 h at 4 °C.Beads were washed extensively, and bound proteins were resolved by SDS-PAGE and detected by Western blotting using anti-His antibody.

结构域截断与点突变互作

Truncation mutants of Protein A and point mutants within the key binding domain were generated by site-directed mutagenesis.Wild-type or mutant plasmids were co-transfected into cells, and co-immunoprecipitation was performed as described above.The interaction intensity was compared between wild-type and mutant groups to identify the critical amino acids responsible for the interaction.

你的细胞系是哪个？（293T？HeLa？还是 HCT116？）