What is Protein Ladder Western Blot?

Western blotting is a powerful tool in molecular biology that allows researchers to detect specific proteins in a sample. However, the process of preparing protein samples for western blotting requires careful attention to detail and precise steps to ensure optimal results. In this article, we will discuss the importance of using protease inhibitors, proper cell lysis, and storage of protein samples to achieve successful Western blots.

Protease Inhibitors: The Key to Preserving Protein Integrity

Proteases are enzymes that can degrade proteins, leading to inconsistent or unreliable results. To prevent this, researchers use protease inhibitors to temporarily inactivate these enzymes. PMSF (phenylmethylsulfonyl fluoride) is a commonly used protease inhibitor with a working concentration of 1 mM. Other protease inhibitors may be selected based on the experimental requirements.

Proper Cell Lysis and Protein Extraction

Cell lysis is a critical step in Western blotting, as it allows researchers to release proteins from their cellular compartments. Centrifugation at 2500 rpm for 3 min followed by cell washing and lysis procedures can help achieve this goal. For cells grown in suspension, centrifugation can be performed at 12000 rpm for 30 sec.

Protein Quantification and Sample Preparation

After protein quantification, researchers add an appropriate amount of 6 × sample loading buffer to the protein samples. The samples are then boiled at 95℃ for 5 min, followed by centrifugation at 12000 rpm for 30 sec. Finally, the samples are stored at -20℃.

Sonication and Cell Lysis

For intracellular suspension of 1mL, sonication is recommended for 10-25 cycles using a Scientz JY92-IIN sonicator with 10% power (650 W). It is essential to avoid touching the tube wall or bottom during sonication. The collected cells can also be fully lysed by sonication.

Protease Inhibitor Addition and Storage

Protease inhibitors should be added before use, as their half-life in water is extremely short. The most commonly used protease inhibitor is PMSF, but other inhibitors may be selected based on the experimental requirements. Protein samples should be stored at -80℃ to avoid repeated freezing and thawing.

In conclusion, preparing protein samples for Western blotting requires careful attention to detail and precise steps to ensure optimal results. By using protease inhibitors, proper cell lysis, and storage of protein samples, researchers can achieve successful Western blots. Additionally, choosing the appropriate method to maximize extraction of target proteins and deciding the appropriate extraction method based on individual protein characteristics are crucial for obtaining reliable results.

What is protein ladder western blot