Hormone Level Detection by ELISA

Serum: Allow whole blood collected in serum separation tubes to stand at room temperature for 2 hours or overnight at 4°C, then centrifuge at 1000×g for 20 minutes. Collect the supernatant, or store it at -20°C or -80°C, avoiding repeated freeze-thaw cycles.

Tissue Homogenate: Rinse the tissue with pre-chilled PBS (0.01M, pH=7.4) to remove residual blood (lysing red blood cells in the homogenate can affect the results). After weighing, mince the tissue. Combine the minced tissue with an appropriate volume of PBS (typically at a weight-to-volume ratio of 1:9, e.g., 1g of tissue sample corresponds to 9mL of PBS; adjust the volume as needed based on the experiment and keep a record. It is recommended to add a protease inhibitor to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, sonicate the homogenate or subject it to repeated freeze-thaw cycles. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for testing.

Cell Lysis: Gently wash adherent cells with pre-chilled PBS, then digest with trypsin. After centrifugation at 1000×g for 5 minutes, collect the cells; suspension cells can be collected directly by centrifugation. Wash the collected cells with pre-chilled PBS three times. Resuspend the cells in 150-200uL of PBS per 1×10^6 cells (add a protease inhibitor to the PBS; if the content is very low, you can appropriately reduce the volume of PBS) and lyse the cells by repeated freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for testing.