When the sample is processed for more than 1 h, it needs to be added once more.į. The rate of loss of activity increases with the increase of pH value, and the deactivation rate at 25℃ is higher than 4℃. PMSF is unstable in aqueous solution, usually it degrades by half in 30 min. However, when the target protein is tightly bound to the genome, the gel needs to be ultrasonically disrupted or syringe-sucked, then take supernatant for subsequent experiments to avoid protein loss.Į. The transparent gel is a genomic DNA component. A viscous transparent gel may appear in the lysate. Because it may introduce protein impurities or cause damage to some certain proteins, especially the membrane surface proteins, to interfere the experimental results.ĭ. It is not recommended to use protease to digest and collect cells. For drug-treated cells, especially samples from apoptosis related studies, media supernatants should also be collected.Ĭ. For the cells grown in suspension, collect by centrifugation at 2500 rpm for 3 min, followed by cell washing and lysis procedures.ī. After protein quantification, add appropriate amount of 6 × sample loading buffer, and boil at 95℃ for 5 min, then centrifuge at 12000 rpm for 30 sec, lastly, store at -20℃.Īll steps must be operated at low temperature! Low temperature! Low temperature!Ī. Be careful not to absorb impurities such as lipids floating in the upper layer. Gently aspirate the supernatant to another fresh tube and place on ice for later use. Basically, the intracellular suspension of 1mL should be sonicated for 10-25 cycles.Į. The sonicator we used is Scientz JY92-IIN, with 10% power (650 W), over 2 sec, stop for 3 sec. Place the ultrasound probe in the middle of the sample lysate, but do not touch the tube wall or tube bottom for ultrasound. The collected cells can also be fully lysed by sonication. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a 1.5 mL EP tube, then place the tube on ice. Add 1 mL of protein lysate containing protease inhibitor to a 10 cm culture dish, shake gently, and lyse on ice for 15-30 min.ĭ. Its half-life in water is extremely short, so it should be added before use.Ĭ. The most commonly used protease inhibitor is PMSF (working concentration is 1 mM), which is highly toxic, so it should be self-protected when used. Appropriate protease inhibitors should be selected according to the experimental requirements. The commonly used protease inhibitors are shown in the table below ( Table 2). Prepare lysates which containing protease inhibitors. After the cell confluence reaches 80%, place the cell culture dish on ice and wash the cells with ice-cold PBS for 3 times.ī. Store Protein samples at -80℃, avoid repeated freezing and thawing, detect as soon as possible.Ī. Choose the appropriate protein lysate to maintain protein solubility. Perform under low temperature and add protease inhibitors to prevent protein degradation.ĭ. Use the appropriate method to maximize the extraction of target protein.Ĭ. Decide the appropriate extraction method based on the characters of individual protein.ī. Generally, complex protein components are extracted from animal or plant tissues or cells, and the following principles should be observed during the extraction process:Ī.

Western blot results