1. Organizing materials: weighing tissue blocks Cheap Pop Up Gazebo,Pop Up Gazebo,Pop Up Gazebo Tent,Steel Frame Gazebo Garden SHANDONG CAIFENG INTERNATIONAL TRADING Co., Ltd. , https://www.zihcaifeng.com
2. Use liquid nitrogen and mortar to crush tissue blocks
3. Add RIPA buffer (3 ml RIPA per gram of tissue), PMSF (30 μl per gram of tissue, 10 mg / ml PMSF), and use Polytron to further homogenize (15,000 rpm * 1 minute) to maintain 4 °
4. Add PMSF (30 μl per gram of tissue, 10 mg / ml PMSF) and incubate on ice for 30 minutes
5. Transfer to a centrifuge tube at 4 ° C, about 20,000 g (about 15,000 revolutions) for 15 minutes
6. The supernatant is cell lysate and can be stored separately in -20 ℃
7. Perform Bradford colorimetry to determine protein concentration
8. Take the same quality cell lysate (volume * protein concentration) and add an equal volume of 2 × electrophoresis sample buffer
9. 3 minutes in boiling water bath
10. Loading
11. Electrophoresis (concentration gel 20mA, separation gel 35mA)
12. Transfer film with electric transfer film instrument (100mA for 40 minutes)
13. The membrane is dyed with Ponceau Red, and the glue is dyed with Coomassie Brilliant Blue
14. Westernblot kit color development
15. Analysis and comparison records