Protein sodium dodecyl sulfate polyacrylamide gel electrophoresis loading buffer

Before plasmid transfection, HEK293T cells were divided into five 10-cm Petri dishes (Corning, USA) and cultured for 1 day. Thereafter, the cells were co-transfected with the myc-tagged protein expression plasmids and flag-tagged plasmids, with pCMS-flag used as a control. After 24 h, the cells were harvested using PBS, the mixture was centrifuged at low speed, the supernatant was discarded, and the pellet was lysed lysed with cell lysis buffer (Beyotime, China). Next, 20 μL of the cell lysate was put aside as an input sample, and the remaining lysate sample was added to anti-flag M2 magnetic beads (Sigma). The mixture was shaken gently at 4°C for more than 2 h, following which the magnetic beads were rinsed three times using cell lysis buffer. The 20 μL input sample and co-immunoprecipitation (co-IP) sample (the rinsed magnetic beads) were then incubated with 2× protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer (Takara) at 100°C for 5 min. The target proteins were examined with the western blot assay using the anti-myc antibody (Transgen, China) and anti-flag antibody (Sigma).

HEK293T cells were grown to an appropriate density in 10 cm dishes (Corning, USA). Afterwards, the cells were co-transfected with 5 μg Myc-tagged protein expression plasmid and 5 μg FLAG-tagged plasmid (pCMS-FLAG as a control). Twenty-four hours after transfection, the cells were harvested and lysed using a cell lysis buffer (Beyotime, China). Next, 20 μL of cell lysate was isolated as an input sample to detect protein expression. An appropriate amount of anti-FLAG M2 magnetic beads (Sigma-Aldrich) was added to the remaining lysate samples. The mixture was incubated at 4°C for > 2 h with gentle shaking, after which the beads were rinsed 3 times with cell lysis buffer. Twenty microliters of input samples and rinsed magnetic beads were then mixed with 2× protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer (Takara) at 100°C under denaturation for 5 min. Western blot assays were employed for target protein examination using an anti-Myc antibody (Abbkine Scientific Co., Ltd., Wuhan, China) and an anti-FLAG antibody (Sigma).

HEK293T cells were divided between two or more Petri dishes (10-cm diameter, Corning) and cultured for 24 h. Fused pCMV-Myc plasmids were co-transfected with vectors expressing FLAG-tagged fusion proteins or empty FLAG vector (control). After 24–36 h, cells were harvested in cell lysis buffer (Beyotime, China). Input samples were prepared from the cell lysate, and the remaining lysates were mixed with anti-FLAG M2 magnetic beads (Sigma, USA) under gentle shaking on a roller at 4°C for 2–4 h. The beads were then washed three times with cell lysis buffer. Input and Co-IP samples were incubated with 2× protein sodium dodecyl sulfate polyacrylamide gel electrophoresis loading buffer (Takara) at 100°C for 3–5 min. Proteins were analyzed by western blotting using anti-Myc antibody (Roche, Switzerland) and anti-FLAG antibodies (Sigma).