What is a phosphorylated antibody?
Antibodies produced against a phosphorylation site in the state of phosphorylation and dephosphorylation of a substrate, so that the phosphorylation and dephosphorylation of the substrate and the change in antibody binding type are used to investigate whether the substrate is phosphorylated, thereby studying changes in protein activity. After the immunogen is phosphorylated, the antibody prepared with it is called phosphorylated antibody.
The significance of phosphorylated antibody
Post-translational modifications are essential for protein biological functions and play an important role in many key regulatory mechanisms of life phenomena. Among them, protein phosphorylation is one of the most common important post-translational modifications. It participates in almost all cellular activities in the body, it is closely related to many biological problems such as signal transduction, cell cycle, proliferation and metabolism, growth and development, and cancer mechanisms. Phosphorylation is usually the addition of phosphate (PO4) groups to proteins under the action of kinases, most commonly on serine (S), threonine (T) or tyrosine (Y) residues. Since phosphorylated proteins can play a complex role, accurate and precise quantification of phosphorylated proteins at the cellular level is essential for deciphering and understanding cellular functions.
The difference between phosphor specific antibodies and ordinary antibodies
- The detection of phosphorylated antibody is based on the protein in an active state (phosphorylation).
- Phospho-antibodies are only designed for phosphorylation sites. It is a site-specific polyclonal antibody, which has some characteristics of monoclonal antibodies.
- Synthesis of common antibody (polyclonal antibody): prokaryotic expression protein-> purification-> immunize animal-> harvest antibody.
- Synthesis of Phosphorylated antibody (phosphor specific antibodies): artificial synthesis of peptides containing phosphorylation sites-> artificial phosphorylation in vitro -> link haptens-> immunize animals-> harvest antibodies.
- A large part of phosphorylated proteins are transcription-related factors. Therefore, phosphorylated antibodies and common antibodies are used to detect the same protein, in situ detection may have different cell localizations. Phosphorylated transcription factor moves from the cytoplasm into the nucleus.
- In general, the experimental results of phosphorylated proteins are more directly related to molecular functions. Ordinary antibodies measure the content of the protein and cannot know its active state. Phosphorylated antibodies measure the level of phosphorylation of the protein, focusing on understanding an active state of the protein.
- Phosphorylated antibodies are mainly prepared for phosphorylation sites, and detect the protein's changes when the cells are stimulated (whether the phosphorylation level is increased or decreased). Ordinary antibodies (mainly prepared for peptides at non-phosphorylated sites) mainly detect the expression of the protein in tissues, cells, tissue distribution, or localization in cells, and can be used as an internal reference for changes in phosphorylation levels.
The application of phosphorylated antibodies
- The level of protein phosphorylation is often related to protein activity: many signaling pathway proteins are caused by a series of cellular responses by changing their phosphorylation level after certain stimulation, but their protein content may not change much.
- Phosphorylated antibody: Prepared for specific phosphorylation sites, and detect changes in the protein's functional state (enhanced or weakened) when the cell is stimulated.
- Non-phosphorylated antibody: It mainly detects the expression of the protein, tissue distribution or cell localization in tissues or cells, and can be used as an internal reference for changes in phosphorylation level.