How do you determine if a protein is phosphorylated?
How do you determine if a protein is phosphorylated?
Methods for Detecting Protein Phosphorylation
- Introduction.
- Kinase Activity Assays.
- Phospho-Specific Antibody Development.
- Western Blot.
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Cell-Based ELISA.
- Intracellular Flow Cytometry and ICC/IHC.
- Mass Spectrometry.
How do you know if a site is phosphorylated?
Identification of Phosphorylation-site involves enriching phosphorylated peptides followed by mass spectrometry. The enrichment is critical since phosphorylated proteins often represent 1-2% of the total protein population.
How do you trace phosphorylation?
Researchers use several techniques for detecting and quantifying protein phosphorylation, including kinase activity assays, phospho-specific antibodies, Western blot, enzyme-linked immunosorbent assays (ELISA), cell-based ELISA, intracellular flow cytometry, mass spectrometry, and multi-analyte profiling.
How do phospho-specific antibodies work?
Phospho-specific antibodies detect only the phosphorylated forms of proteins in a complex protein mixture within cells. They recognize the phosphorylated amino acids in the context of the surrounding amino acid sequence.
What residues can be phosphorylated?
Phosphorylation can occur on serine, threonine and tyrosine side chains (often called ‘residues’) through phosphoester bond formation, on histidine, lysine and arginine through phosphoramidate bonds, and on aspartic acid and glutamic acid through mixed anhydride linkages.
How does phosphorylation affect SDS PAGE?
The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE.
Where does phosphorylation occur?
Substrate-level phosphorylation occurs in the cytoplasm of cells (glycolysis) and in the mitochondria (Krebs cycle). It can occur under both aerobic and anaerobic conditions and provides a quicker, but less efficient source of ATP compared to oxidative phosphorylation.
Can mass spectrometry identify phosphorylation sites?
High resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity and throughput. One can also IP only phospho-tyrosine peptides using a pTyr antibody followed by LC-MS/MS.
What is phospho antibody?
Phosphospecific antibodies are affinity-purified rabbit polyclonal or monoclonal antibodies that are monospecific for a target protein that is phosphorylated. These antibodies provide critical insight into the investigation of complex signal transductions events.
How are phospho antibodies made?
Phospho-specific antibodies are generated using peptides containing one or more phosphorylated amino acids. There are three residues which can be phosphorylated: Serine (S), Threonine (T) and Tyrosine (Y).
What are the 3 types of phosphorylation?
Three of the most common forms of phosphorylation are:
- Glucose phosphorylation.
- Protein phosphorylation.
- Oxidative phosphorylation.
Does phosphorylation activate or deactivate?
The phosphorylation of a protein can make it active or inactive. Phosphorylation can either activate a protein (orange) or inactivate it (green). Kinase is an enzyme that phosphorylates proteins. Phosphatase is an enzyme that dephosphorylates proteins, effectively undoing the action of kinase.
