The concentration of the Oriscience protease inhibitor cocktail should be determined based on the experimental type, sample type, and characteristics of the target protein. The core principle is to effectively inhibit protein degradation while avoiding interference with experimental results. This mixture is usually provided as a concentrate and must be diluted to the working concentration. This process requires precise calculation considering the lysis buffer volume, sample volume, and the inhibitor's mechanism of action.
For protein extraction from routine mammalian cell or tissue samples, Oriscience recommends adding the protease inhibitor cocktail to the lysis buffer at a ratio of 1:100. For example, if using 1 mL of lysis buffer to lyse cells or tissue, add 10 μL of the 100× concentrated inhibitor mixture. This concentration covers most experimental scenarios, effectively inhibiting the activity of common endogenous proteases such as serine and cysteine proteases, preventing target protein degradation during extraction. If the experiment involves special sample types, such as plant tissues or microbial cells, the concentration may need to be adjusted according to the type and activity of the proteases in the sample. For example, plant samples may contain more active cysteine proteases; in this case, the ratio of the inhibitor mixture can be increased to 1:50 or higher to ensure sufficient inhibition.
The type of experiment significantly influences the choice of inhibitor concentration. In experiments requiring high protein integrity, such as Western blotting or immunoprecipitation, recommended concentrations must be strictly followed to avoid target protein degradation due to insufficient inhibitor concentration or adverse antibody binding efficiency due to excessive concentration. However, in experiments requiring partial protease activity, such as studying substrate specificity or regulatory mechanisms of proteases, the inhibitor concentration can be appropriately reduced, or even a single inhibitor can be used instead of the mixture, to achieve precise control of specific protease activity.
The characteristics of the target protein are another key factor in determining the inhibitor concentration. If the target protein is sensitive to proteases, such as proteins easily cleaved by cysteine proteases, the concentration of the corresponding component in the inhibitor mixture must be sufficiently high. Oriscience protease inhibitor cocktails typically contain multiple components, such as leupeptin, pepstatin A, and aprotinin, each targeting different types of proteases. By consulting product instructions or relevant literature, one can understand the target protease type and recommended working concentration of each component, thereby adjusting the overall concentration of the mixture or supplementing with specific inhibitors based on the characteristics of the target protein.
The storage conditions and usage of the inhibitor mixture also affect its effective concentration. The concentrate must be stored at -20°C to maintain component stability. Before use, it must be completely thawed and agitated to avoid uneven concentration due to component precipitation. After adding to the lysis buffer, it must be thoroughly mixed and used immediately to avoid decreased inhibitor activity due to prolonged storage. If the experiment requires long-term lysis or multiple freeze-thaw cycles, consider adding a higher concentration of the inhibitor mixture to the lysis buffer, or adding the inhibitor stepwise during lysis to ensure continuous inhibition of protease activity.
In experiments involving phosphorylated proteins, it is also necessary to consider whether to add a phosphatase inhibitor simultaneously. Oriscience protease inhibitor cocktails typically do not contain phosphatase inhibitors. If phosphatase activity inhibition is required, a phosphatase inhibitor mixture must be added separately and mixed with the protease inhibitor mixture and lysis buffer at the recommended ratio. In this case, the protease inhibitor mixture should still be added at a concentration of 1:100, while the phosphatase inhibitor ratio should be adjusted according to the product instructions to ensure simultaneous inhibition of both protease and phosphatase activity, protecting the integrity and phosphorylation state of the target protein.
