We usually use the pospho Protein: total protein ratio to evaluate the protein phosphorylation. But what does it mean if we take total protein: pospho protein ratio? Because when I take this ratio I get a nice correlation.
If you have a nice correlation using the total protein: phospho protein ratio you should get the same nice correlation with the reversed ratio.... but the phospho Protein: total protein is more informative: total protein is roughly constant whereas phosphorylated protein can varies according to physiological conditions. Division by the total amount of protein is just used to account for (usually small) experimental fluctuations in lysate recovery.
Hi Arezou Abdi
The total protein: phospho protein ratio in Western blot analysis refers to the ratio of the total amount of protein (usually determined by staining with a protein-specific dye) to the amount of phosphorylated protein (detected by an antibody against a specific phosphorylated site). The ratio provides insights into the relative phosphorylation status of a protein within a sample.
Typically, researchers use the phospho protein: total protein ratio to assess protein phosphorylation because it helps normalize the phosphorylation levels to the overall protein expression levels. This normalization accounts for any differences in protein loading or expression between samples and allows for more accurate comparisons.
However, if you are obtaining a nice correlation with the total protein: phospho protein ratio, it suggests that there may be an interesting relationship or association between the total protein levels and the phosphorylation status. It's important to note that the interpretation of this correlation would depend on the specific context and biological question being addressed in your study.
The total protein: phospho protein ratio alone does not provide direct information about the specific phosphorylation events occurring on the protein or the functional consequences of those phosphorylations. It is still important to validate and interpret the results in conjunction with other relevant data, such as protein localization, downstream signaling effects, and functional assays, to gain a comprehensive understanding of the phosphorylation events and their biological significance.
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