Throughout the sample-preparation process, freeze/thaw steps and homogenization facilitate enzymatic activity by providing access to substrates not normally encountered by enzymes, thereby accelerating degradation. Denator has developed a system that eliminates all enzymatic activity (Figure 1).
Use of the Stabilizor™ T1 (Figure 2), which is based on rapid and uniform thermal inactivation, leads to denaturation and permanent inactivation of enzymes. This is accomplished by exposing the sample to conductive heating, assisted by vacuum suction and pressure during a specific period of time—this ensures efficient and complete stabilization of the sample.
Stabilizor T1 measures the sample with a laser and automatically assesses the optimal treatment for each sample, guaranteeing reproducibility. Frozen samples can be stabilized as is, thus preventing the massive proteolytic degradation that occurs during thawing. Stabilized samples can be analyzed using all major protein study workflows and standard protocols with only minor adjustments.
The addition of chemical enzyme inhibitor cocktails or manipulations such as cross-linking or pH alterations are common means of minimizing post-sampling degradation. Rapid and homogeneous thermal inactivation has advantages over other methods that are either reversible, added during later stages, not suitable for specific downstream analysis techniques, or impractical or limited in some other way, e.g., toxic or expensive.
To demonstrate the efficiency of the Stabilizor system, phosphatase activity was compared in protein extracts from stabilized and nonstabilized mouse brains, extracted with and without phosphatase- and protease inhibitors. The results show high activity in extracts from nonstabilized brain. Roughly half of the activity remained despite the presence of inhibitors during extraction. In stabilized samples, activity levels were detected at background levels, thereby showing that enzymatic activity is eliminated following Stabilizor treatment and that there is no further risk of enzymatic degradation or changes occurring in stabilized samples (Figure 3).
In addition, Western blot analysis of phosphorylated CREB, GSK3b, and ERK1/2, show that stabilized samples conserve phosphorylations for up to two hours at room temperature. When the same proteins were analyzed after only 10 minutes post-mortem in snap frozen samples, they were detected at significantly lower levels, indicating extensive ex vivo dephosphorylation.