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Preserve phosphorylation states
An ability to determine phosphorylation states that reflect the in vivo status as closely as possible is crucial to understanding the role of phosphoproteins in diseases such as cancer, neurodegeneration and atherosclerosis, in disease prognosis and in determining the action of a potential therapeutic. However, phosphorylation states are highly sensitive to disturbances and can change rapidly due to kinase and phosphatase activity that may continue after sampling.
Maintain phosphorylation states post-sampling
An evaluation of heat stabilization technology compared to conventional snap freezing, demonstrated that, during 30min at room temperature, phosphorylation states continued to change in snap-frozen samples of hippocampal lysates.
Phosphorylation states remain constant in heat-stabilized samples
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Histograms represent average values for each group of samples relative to 100% for snap-frozen samples at 0 min RT (white); 30 min RT (grey); heat-stabilized samples at 0 min RT (black); 30 min RT (stripes). Statistical significance determined by unpaired Student’s t-test.
Results courtesy of Ahmed et al. Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization. Journal of Neuroscience Methods, 2011 March
Permanently inactivate kinase and phosphatase activity without the use of inhibitors
Heat stabilization abolished 99.6% of kinase activity
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Only three out of 144 peptides produced measurable fluorescence in heat-stabilized samples. |
Results courtesy of Harvard Medical School (Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis. Smejkal et al., Electrophoresis 2011, Volume 32, pages 2206–2215).
Heat stabilization permanently inactivates phosphatases
Phosphatase activity measured using SensoLyte pNPP phosphatase kit (Anaspec).
Heat-stabilized mouse brain cortex samples show a significant lower level of phosphatase activity compared to snap frozen samples with and without inhibitors. Phosphatase activity is equivalent to background levels (Svensson et al. Heat stabilization of the Tissue Proteome: A New Technology for Improved Proteomics. J Proteome Res. 2009)
