Production of a site- and phosphorylation state-specific antibody
- 18 October 2007
- journal article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 2 (10), 2574-2581
- https://doi.org/10.1038/nprot.2007.374
Abstract
Protein phosphorylation plays important roles in various aspects of cellular events. Visualization of site-specific phosphorylation in cells is of great importance not only to analyze spatial and temporal distribution but also to investigate biological function. Now, site- and phosphorylation state-specific antibodies are widely utilized as the most powerful tools for these analyses. This protocol details a method to produce the polyclonal version of such an antibody by immunizing a synthetic phosphopeptide corresponding to a protein phosphorylated at targeted site(s). This protocol is also applicable to the production of other types of antibodies, which specifically recognize the site-specific modification, such as acetylation, methylation and proteolysis. The protocol can be completed in 2-3 months.Keywords
This publication has 21 references indexed in Scilit:
- A decade of site‐ and phosphorylation state‐specific antibodies: recent advances in studies of spatiotemporal protein phosphorylationGenes to Cells, 2001
- The DNA damage response: putting checkpoints in perspectiveNature, 2000
- Rho GTPases and their effector proteinsBiochemical Journal, 2000
- A Long Twentieth Century of the Cell Cycle and BeyondCell, 2000
- Protein Phosphorylation and Signal TransductionPharmacology & Therapeutics, 1999
- Intracellular Signaling by Hydrolysis of Phospholipids and Activation of Protein Kinase CScience, 1992
- A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: Application to a non-radioactive method for measuring protein kinase activitiesBiochemical and Biophysical Research Communications, 1991
- [23] Production of phosphorylation state-specific antibodiesMethods in enzymology, 1991
- [11] Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose platesMethods in enzymology, 1991
- Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.Proceedings of the National Academy of Sciences of the United States of America, 1983