Protein phosphorylation can best be assessed by:
Evaluation of protein phosphorylation status demands method preserving labile phosphoester bonds and providing specificity for phosphorylated residues. Western blotting accomplishes this by resolving lysates via SDS-PAGE, transferring to PVDF, and immunoblotting with phospho-specific antibodies that recognize phosphoserine, phosphothreonine, or phosphotyrosine in context of surrounding amino acids. Comparison with total protein antibody after stripping reveals stoichiometry. Southern blotting and restriction fragment length polymorphism analyze DNA polymorphisms, while chromatin immunoprecipitation evaluates DNA-protein occupancy, none measuring covalent phosphate addition directly, making phospho-Western the preferred validated assay for signaling research.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.