Optimize protein integrity and yields during workflows
Inside a cell, proteins are commonly separated from proteolytic enzymes through differential localization. Disruption of cellular and tissue architecture during protein extraction distorts the in vivo state by making all proteins potentially accessible for degradation or modification by endogenous proteases and phosphatases. Unregulated, these activities can reduce protein yield and function in a sample. To minimize these losses, protease and phosphatase inhibitors can be added during cell lysis to prevent degradation of extracted proteins and preserve protein structure, function, and yields during sample preparation.
Protease and phosphatase inhibitors
Protease and phosphatase inhibitor cocktails and tablets are ideal for protecting proteins during extraction procedures or lysate preparation using primary cells, cultured mammalian cells, animal tissues, plant tissues, yeast cells, or bacterial cells. Formulations are packaged in multiple sizes, and EDTA-free versions are available for divalent cation–sensitive assays. Thermo Scientific Pierce inhibitor mini tablets have been reformulated to dissolve quickly into a clear solution and are fully compatible with all Pierce protein assays. Figures 1 and 2 provide representative examples of the effectiveness of Pierce Protease and Phosphatase Inhibitor Mini Tablets.
Figure 1. Performance comparison between three commercially available protease inhibitor tablets. Pancreatic extract (100 μL, 0.5 μg/μL) was incubated with cleavable fluorogenic substrates for trypsin and cysteine proteases, metalloproteases, and cathepsins in the presence of Thermo Scientific Pierce Protease Inhibitor Mini Tablets, Roche cOmplete Protease Inhibitor Tablets, and Sigma-Aldrich SIGMAFAST Protease Inhibitor Cocktail Tablets, with and without EDTA. Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the appropriate detection emission wavelengths on a Thermo Scientific Varioskan Flash microplate reader. Percent protease inhibition is shown for each protease inhibitor formulation.
Figure 2. Inhibition of phosphatase activity in human colorectal carcinoma cell lysate. HCT116 cells were serum-starved and treated with epidermal growth factor (EGF) for 15 min or left untreated as controls (data not shown). Cell lysates were prepared in Thermo Scientific Pierce IP Lysis Buffer with or without Thermo Scientific Pierce Protease and Phosphatase Inhibitor Mini Tablets, EDTA-Free and incubated with anti–phosphotyrosine antibody overnight at 4ºC. The immunoprecipitated complex was incubated with Thermo Scientific Pierce Protein A/G Magnetic Beads for 1 hr at room temperature. After washing the beads, a low-pH elution was performed, and this eluate was run on a gel, transferred to a membrane, and probed with anti– EGF receptor (EGFR) antibody prior to chemiluminescence western blot detection.
Download the free Protein Preparation Handbook
The Protein Preparation Handbook provides technical information on our broad portfolio of reagents and tools for protein extraction, cleanup, immunoprecipitation, and purification, including the inhibitor tablets discussed here. Practical information, selection guides, and relevant data are included to help improve protein yield, facilitate downstream analysis, and optimize workflows.
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