1. Sample Collection
A. Serum
1000 after natural agglutination of whole blood samples for 30 minutes × Centrifuge for 15 minutes and take clarification to detect. Clarification can be stored at -20 ℃ to avoid repeated freeze-thaw.
B. Plasma
EDTA or heparin can be used as Anticoagulant, and within 30 minutes after specimen collection, 1000 at 2-8 ℃ × Centrifuge for 15 minutes or place the specimen at -20 ℃ to avoid repeated freeze-thaw cycles. (Note: Hemolysis in the sample can affect the final test result, so hemolytic samples should not be subjected to this test)
C. Cell supernatant
Collect cell culture medium, centrifuge the supernatant, and store at -20 ℃ to avoid repeated freezing and thawing.
D. Cell lysis sample
1. After collecting cells, wash 3 times with pre cooled 10 mM PBS, 5000 × Centrifuge for 5 minutes.
2. Cell count, centrifuge and discard the supernatant;
3. Add PMSF to the cell lysate, with a final concentration of 1mM;
4. Press every 1 × 107 cells were added with 1ml of cell lysis solution (including PMSF) and subjected to ice lysis for 30 minutes. During this time, the cells were inverted to make the lysis more complete, and subjected to ultrasonic fragmentation treatment;
5. 4 ℃, 10000 × Centrifuge for 10 minutes;
6. Detection of total protein concentration in cell lysis samples;
7. The entire process needs to be operated on ice to prevent protein degradation.