Protein a g sepharose

Cells were plated in 10-cm dishes. Cell monolayers were lysed using NP40 and lysates were centrifuged at 4000×g for 20 min. Then, 60 μl of Protein A/G Sepharose (P2012; Beyotime Biosciences) was added to the lysate, blocked for 2 h, then centrifuged at 2000 rpm to remove the magnetic beads. The remaining protein lysate was divided into two parts, and 5 µg target antibody and anti-mouse/rabbit IgG were added to each part and incubated overnight at 4 °C while shaking in a chromatography cabinet. The next day, 25 μl agarose A/G magnetic beads were added to each tube and incubated at 4 °C for 6 h; the cell lysate was then washed with lysis buffer and the tubes were heated in boiling water for 10 min. The samples were then used for western blotting.

The desired cell line was plated in two 10‐cm cell culture dishes. After the cells grew to confluence, they were lysed and centrifuged at 12 000 rpm for 15 minutes at 4℃. Next, 60 μL of Protein A/G Sepharose (P2012; Beyotime Biosciences) was added to the supernatant and stirred for at least 2 hours. The mixture was then centrifuged at 4°C and 1000 rpm for 5 minutes, following which the supernatant was divided into two parts. To one part was added the target antibody (8 µg), and to the other part, anti‐mouse/rabbit IgG (1:2000; ZSGB‐BIO, Beijing, China). The mixture was shaken overnight at 4°C in a chromatography cabinet. The following day, 25 μL of agarose A/G magnetic beads was added to each tube and incubated at 4°C for 6 hours. Next, the cell lysate was washed, heated in boiling water for 10 minutes and immunoblotted.

Rat spinal cord of lumbar vertebra was collected and mitochondrial fraction was extracted as described above. Approximately 100 μg from the mitochondrial fraction were used for coimmunoprecipitation. The protein sample was mixed with 20 μl protein A+G sepharose (Beyotime, Shanghai, China) and incubated for 30 min at 4°C. Then the samples were centrifuged at 12,000 g for 10 min. The supernatant was incubated with 2 μg polyclonal rabbit anti-Bcl-xL antibody (1:1000) and 15 μl of protein A+G sepharose (50% slurry) for 5 h at 4°C. After centrifugation at 12,000 g for 1 min, the supernatant was subjected to Western blot analysis with anti-Bad antibody (1:500).

The cell lines used in the experiment were seeded in two 10-cm cell culture dishes. When the cells reached confluence, they were lysed for 20 min and centrifuged at 12000 rpm for 15 min at 4 °C. Next, 40 μL of protein A/G Sepharose (P2012; Beyotime Biosciences) was added to the supernatant and blocked for at least 2 h. The mixture was then centrifuged at 1000 rpm for 5 min at 4 °C, and the supernatant was divided into two parts. Anti-ACAT1 or anti-FUS antibody (8 μg) was added to one part, and anti-mouse/rabbit IgG (1:2000; ZSGB-BIO, Beijing, China) to the other part. The mixture was shaken overnight at 4 °C. The next day, 25 μL of agarose A/G magnetic beads was added to each tube and incubated at 4 °C for 6 h. The cell lysate was then washed, heated in boiling water for 10 min, and finally, immunoblotting was performed.

The cell line of interest was plated in two 10-cm dishes. When 100% confluency was reached, the cells were lysed and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected for Co-IP, and 60 μL Protein A/G Sepharose (P2012; Beyotime Biosciences) was added to the supernatant to block for 2 h; the supernatant was then centrifuged at 1000 rpm for 5 min at 4 °C and divided into two parts on average; 4–10 μg of the target antibody and anti-mouse/rabbit IgG (1:2000; ZSGB-BIO, Beijing, China) were added, and the antibody was shaken overnight in a 4 °C chromatography cabinet. On the next day, 25 μL of agarose A/G magnetic beads were added to each tube and incubated at 4 °C for 6 h, after which the cell lysate was washed with lysis buffer, and the tubes were heated in boiling water for 10 min followed by immunoblotting.

The spinal cords of rats were collected and mitochondrial fraction was extracted. Approximately 100 μg mitochondrial fraction was used for coimmunoprecipitation. The protein sample was mixed with 20 μl protein A+G sepharose (Beyotime, Shanghai, China) and incubated for 30 min at 4 °C, and then the samples were centrifuged at 1,200 × g for 10 min. The supernatant was incubated with 2 μg polyclonal rabbit anti-Bcl-xL antibody (1:1000) and 15 μl of protein A+G sepharose (50% slurry) for 5 h at 4 °C. Protein A + G beads were collected by centrifugation at 1,200 × g for 5 min and were washed 6 times with TNE buffer (10 mM Tris-HCl at pH 7.5, 1% NP-40, 0.15 M NaCl, 1 mM EDTA and 1:100 protease inhibitor cocktail). Bound proteins were eluted in sample buffer, separated on 4–12% SDS-PAGE, and blotted with anti-Bad and anti-Bcl-xL antibodies.