Two-dimensional gel electrophoresis (2-D GE) is a proven tool in the protein study toolbox. To minimize protein degradation, inhibitors are routinely added to the urea-based buffers used for protein extraction. The effect of the Stabilizor system was tested on this relatively degradation-safe system in Professor Michael J. Dunn’s laboratory at the Proteome Research Centre, UCD Conway Institute, Dublin, Ireland.
Mouse brains were harvested and either directly frozen, stabilized before freezing, or stabilized from a frozen state. Large differences were detected between stabilized and nonstabilized samples with a clear trend of fewer low molecular weight (LMW) spots and higher intensities of high molecular weight (HMW) spots in stabilized samples compared to nonstabilized.
Decreased LMW spots were most often subsequently identified as degradation fragments, whereas increased HMW spots were mostly identified as full-length proteins. This indicates that despite the use of state-of-the-art inhibitors and urea buffers, degradation still remains a problem that may lead to misleading results.
A second case study examined phospho-specific immunohistochemistry with the department of pathology and cytology at Uppsala University in Sweden. Protein phosphorylations modulate activity and sub-cellular localization and are frequently involved in cell signaling. Phosphorylation states change rapidly in response to external stimuli and are often drastically affected by post-mortem changes. Formalin, which is routinely used to fixate tissue samples prior to IHC, penetrates tissue slowly, ~0.5–2 mm/h, allowing for extensive changes in phosphorylation states prior to complete fixation.
The stabilization of phosphorylations with the Stabilizor system was evaluated using phosphospecific antibodies, here exemplified with CREB phosphorylated on Ser133 (pCREB(Ser133)). Mouse brains were either directly fixed in formalin or stabilized prior to formalin fixation.
To assess stability of pCREB(Ser133) at room temperature, samples were incubated at room temperature prior to, or after, stabilization. In samples directly subjected to formalin fixation, staining was primarily localized close to the tissue surface, consistent with loss of phosphorylations during formalin penetration.
In stabilized samples, pCREB positive staining could be seen throughout the tissue. In nonstabilized tissue, all traces of pCREB(Ser133) staining were gone after only 15 minutes of incubation at room temperature, whereas stabilized samples showed unchanged levels of staining even after 24 hours at room temperature prior to formalin fixation, indicating complete stabilization of phosphorylations.
The importance of correct sample preparation cannot be overstated. Sample quality is crucial and must be considered throughout the whole experiment. The Stabilizor system eliminates degradation and is compatible with all major protein study workflows. In addition, it enables preservation of the proteome in a biologically relevant state enabling confident biomarker identification.