Preserve phosphorylation states - compare true biological variation

Determining, as closely as possible, the in vivo status of phosphpoproteins is crucial to understanding their role, yet phosphorylation states are highly sensitive to disturbances and can change rapidly. By using heat stabilization immediately after sampling, enzymatic activity is permanently stopped, allowing for comparison of true biological variation.

Heat stabilization abolished 99.6% of kinase activity


Graph shows 18 of the most fluorescent peptides in a 144 peptide array in a kinase activity study. Only three were measurable phosphorylated in heat stabilized samples.
Results derived from 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

Enzymatic activity in snap frozen mouse cortex samples compared to heat stabilized. Phosphatase activity was measured using pNPP phosphatase kit (Anaspec). The phosphatase activity in heat stabilized samples is equivalent to background levels compared to snap frozen samples with and without inhibitors (PhosSTOP and c0mplete, Roche).
Heat stabilization of the Tissue Proteome: A New Technology for Improved Proteomics. Svensson et al., J Proteome Res. 2009

Heat stabilization preserves actual phosphorylation states

Quantification of phosphoproteins in mouse hippocampal lysates. Heat stabilized and snap frozen samples with inhibitors, were analyzed by Western blot immediately or after 30 min in room temperature (RT).
Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization. Ahmed et al., Journal of Neuroscience Methods, 2011

Application note, Global protein phosphorylation analysis with heat stabilized samples

Visit our User support zone to learn more on how to work with heat stabilized samples and view guidelines for several downstream analytical methods and the key parameters important to think about for each workflow.

"At last we have a solution to be able to preserve phosphorylated proteins in clinical samples."
Prof Peter James, CREATE Health, Lund University

"..significantly higher yield of phosphoprotein when compared to standard snap freeze preservation"
Dr. C. Rountree et al, Proteome Science, 2010; 8: 61