response. When serine 337 is phosphorylated by protein kinase A in vitro, the DNA binding efficiency of the p50 subunit of NF-κB is greatly increased. Phosphorylation of serine and threonine residues is known to crosstalk with O-GlcNAc modification of serine and threonine residues. Tyrosine Tyrosine phosphorylation is fast to react and the reaction can be reversed. Being one of the major regulatory mechanisms in signal transduction - cell growth, differentiation, migration and metabolic homeostasis are cellular processes maintained by tyrosine phosphorylation. The function of protein tyrosine kinases and protein-tyrosine phosphatase counterbalances the level of phosphotyrosine on any protein. The malfunctioning of specific chains of protein tyrosine kinases and protein tyrosine phosphatase has been linked to multiple human diseases such as obesity, insulin resistance, and type 2 diabetes mellitus. Phosphorylation on tyrosine doesn't occur in just eukaryotes but has been discovered to occur in a selection of bacterial species and present among prokaryotes. Phosphorylation on tyrosine maintains the cellular regulation in bacteria similar to its function in eukaryotes. Arginine Arginine phosphorylation in many Gram-positive bacteria marks proteins for degradation by a Clp protease. Non-canonical phosphorylation on His, Asp, Cys, Glu, Arg and Lys in human cells Recent studies from Claire E Eyers lab confirm widespread human protein phosphorylation on multiple non-canonical amino acids, including motifs containing phosphorylated histidine (1 and 3 positions), aspartate, cysteine, glutamate, arginine and lysine in HeLa cell extracts. Due to the chemical and thermal lability of these phosphorylated residues, special procedures and separation techniques are required for preservation alongside the heat stable 'classical' Ser, Thr and Tyr phosphorylation. Detection and characterization Antibodies can be used as powerful tool to detect whether a protein is phosphorylated at a particular site. Antibodies bind to and detect phosphorylation-induced conformational changes in the protein. Such antibodies are called phospho-specific antibodies; hundreds of such antibodies are now available. They are becoming critical reagents both for basic research and for clinical diagnosis. Posttranslational modification (PTM) isoforms are easily detected on 2D gels. Indeed, phosphorylation replaces neutral hydroxyl groups on serines, threonines, or tyrosines with negatively charged phosphates with pKs near 1.2 and 6.5. Thus, below pH 5.5, phosphates add a single negative charge; near pH 6.5, they add 1.5 negative charges; above pH 7.5, they add 2 negative charges. The relative amount of each isoform can also easily and rapidly be determined from staining intensity on 2D gels. In some very specific cases,