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.
How is histidine phosphorylated?
On the other hand, histidine undergoes N-linked phosphorylation on either nitrogen on its imidazole ring to form a high-energy phosphoramidate bond (N-P), which is labile at low pH and high temperature (3).
What is a 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 do you detect hyperphosphorylation?
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.
Why do we enrich our sample for Phosphopeptides?
The advantage of phosphoprotein enrichment is that it usually reveals the molecular weight and the isoelectric point of proteins. Such information could be helpful for subsequent protein identification by MS. Another advantage with the phosphoprotein-enriching approach is that intact proteins are separated.
Can aspartic acid be phosphorylated?
Phosphorylation is found most commonly on specific serine and threonine amino acid residues in proteins, but it also occurs on tyrosine and other amino acid residues (histidine, aspartic acid, glutamic acid) as well.
What does phospho stand for?
, phosph- [Fr. phosphorique, phosphoric] Prefixes meaning phosphorus.
What is the role of antibodies in the identification of phosphoproteins?
Mass spectrometry is the gold standard for large-scale identification and quantification of phosphoproteins 3, but antibodies are indispensable for their detection in enzyme-linked immunosorbent assays (ELISA), western blot analysis, immunoimaging, proximity ligation, protein microarrays and flow cytometry, and for affinity purification.
What is the structure of the β-arrestin-1-v2rpp-fab30 complex?
The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the ‘lariat loop’ implicated in maintaining the inactive state of β-arrestin-1.
How are antibody reagents specific to phosphorylated peptides generated?
Antibodies specific to phosphorylated peptides are readily generated through structure-based engineering and in vitro selection. The study of post-translational modification of proteins, a critically important aspect of cell physiology and pathology, depends on the availability of specific antibody reagents.
What is a targettryptic phosphopeptide map?
Tryptic phosphopeptide maps are prepared from kinase subunits that had been autophosphorylated or dephosphorylated for different times. The time course of phosphorylation of a particular site is determined by plotting the fraction of total radioactivity that is recovered in the particular site at each time point.