Phosphorylations

The importance of understanding the role of post-translational modifications (PTMs), such as phosphorylations, in diseases, as drug targets and biomarkers, is well acknowledged. To accurately determine the phosphorylated state of a protein in vivo is crucial to understand its role in disease prognosis and to predict the specificity or toxicity of a potential therapeutic.

Phosphorylated proteins change rapidly post sampling. However, using the Stabilizor T1 system to rapidly inactivate all enzymes preserves phosphorylated proteins at their in vivo levels:

Completely inactivates phosphatase and kinase activity
Phosphorylations remain stable in room temperature
Eliminates need for costly and/or inefficient phosphatase inhibitors

The Stabilizor system has been used to preserve phosphorylations prior to:

Mass spectrometry-based analysis
Western Blot with phospho-specific antibodies
1D and 2D gels with phospho-specific stains
Reversed phased protein arrays (RPPA)

Phosphatase activity was measured using SensoLyte pNPP phosphatase kit (Anaspec). Stabilized mouse brain cortex samples show a significant lower level of phosphatase activity compared to snap frozen samples with and without inhibitors.

Levels of phosphorylated CREB, GSK and MAPK, in samples of mouse brain cortex were stable for up to two hours in room temperature. Same phosphorylated proteins were significantly down-regulated 10 min post-mortem in snap frozen samples.




	Phosphorylations The importance of understanding the role of post-translational modifications (PTMs), such as phosphorylations, in diseases, as drug targets and biomarkers, is well acknowledged. To accurately determine the phosphorylated state of a protein in vivo is crucial to understand its role in disease prognosis and to predict the specificity or toxicity of a potential therapeutic. Phosphorylated proteins change rapidly post sampling. However, using the Stabilizor T1 system to rapidly inactivate all enzymes preserves phosphorylated proteins at their in vivo levels: Completely inactivates phosphatase and kinase activity Phosphorylations remain stable in room temperature Eliminates need for costly and/or inefficient phosphatase inhibitors The Stabilizor system has been used to preserve phosphorylations prior to: Mass spectrometry-based analysis Western Blot with phospho-specific antibodies 1D and 2D gels with phospho-specific stains Reversed phased protein arrays (RPPA) Phosphatase activity was measured using SensoLyte pNPP phosphatase kit (Anaspec). Stabilized mouse brain cortex samples show a significant lower level of phosphatase activity compared to snap frozen samples with and without inhibitors. Levels of phosphorylated CREB, GSK and MAPK, in samples of mouse brain cortex were stable for up to two hours in room temperature. Same phosphorylated proteins were significantly down-regulated 10 min post-mortem in snap frozen samples.

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