Western blotting
The spinal cords were cut into small fragments and added into the
lysates, and a mixture of protein phosphatase inhibitors was added into
the tissue cell lysates at a proportion of 1:100. The cracked samples
were centrifuged for 3-5 minutes at 10,000-14,000×g . The BCA
Protein Quantitation Kit(Bioteke Corporation) was used to draw the
standard curve and calculate the protein concentrations. For each 30µl
protein sample, 10 µl of buffer was added and blended, and proteins were
denatured by heating in a 100°C water bath for 3-5 minutes. After
cooling to 25°C, the samples were centrifuged at 10,000-14,000rpm for
2-5 minutes. The supernatants were collected and directly used for
electrophoresis.