What details should be paid attention to during the experimental process of protease inhibitor test?

In the process of protease inhibitor testing, the following details need to be noted to ensure the accuracy and reliability of the experiment:
Choose the appropriate concentration: When using, the appropriate concentration should be selected according to the experimental needs. Excessive concentration may lead to strong inhibitory effects and affect normal cell function; Low concentration may not have an inhibitory effect. ‌
Control usage time: Protease inhibitors and phosphatase inhibitors should be used for an appropriate amount of time, as prolonged inhibition may cause damage to cells or samples. ‌
Pay attention to the selectivity of inhibition: Some protease inhibitors and phosphatase inhibitors may have inhibitory effects on multiple enzymes, so when selecting, attention should be paid to their selectivity towards the target enzyme and avoid inhibiting other important enzymes. ‌
Storage conditions: Protease inhibitors and phosphatase inhibitors should be stored in a high-temperature and humid environment to avoid deactivation. ‌
Compatibility considerations: If multiple protease inhibitors and phosphatase inhibitors are used simultaneously, attention should be paid to their interactions and possible adverse effects. ‌
Sample processing: During the processing, the sample should be quickly and accurately placed in an ice box as much as possible to avoid sample degradation caused by stale or prolonged exposure to room temperature. ‌
Add enzyme inhibitor to lysis buffer: The ratio of enzyme inhibitor to lysis buffer is generally 1:100, and the principle is to add it as needed. If producing phosphorylated antibodies, phosphatase inhibitors need to be added to the lysis buffer that has already been treated with protease inhibitors, at a ratio of 1:100. ‌
Sample protein concentration: Quantitatively measure the concentration of extracted proteins using BCA, adjust the required protein concentration to 3mg/ml by adding different proportions of loading buffer, and then conduct subsequent electrophoresis experiments. ‌
Following these details can ensure the accuracy and reliability of experimental results, thereby facilitating better research on protease inhibitors. ‌