Side by side comparison of two commercially available anti-biotin polyclonal antibodies with Cell Signaling Technology's anti-biotin (A7C2A) rabbit monoclonal antibody. Mouse liver peptides were chemically biotinylated and 1 μg was mixed with 1 mg of untreated mouse liver peptides for each sample. Immunoprecipitated peptides from each of the three antibodies were then analyzed by OrbiTrap™ LC-MS and the average number of biotinylated peptides identified is shown.
Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.
Biotin, also known as vitamin B7, is a chemical moiety that can be post-tranlationally added to proteins via a process known as biotinylation. Biotinylated proteins and peptides bind very tightly to avidin groups. Biotinylation of protein targets can be achieved using chemical or enzymatic methods.
PTMScan® Technology employs a proprietary methodology from Cell Signaling Technology (CST) for peptide enrichment by immunoprecipitation using a specific bead-conjugated antibody in conjunction with liquid chromatography (LC) tandem mass spectrometry (MS/MS) for quantitative profiling of post-translational modification (PTM) sites in cellular proteins. These include phosphorylation (PhosphoScan®), ubiquitination (UbiScan®), acetylation (AcetylScan®), and methylation (MethylScan®), among others. PTMScan® Technology enables researchers to isolate, identify, and quantitate large numbers of post-translationally modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of PTMs in cell and tissue samples without preconceived biases about where these modified sites occur (1). For more information on PTMScan® Proteomics Services, please visit www.cellsignal.com/services/index.html
Biotinylation of proteins and peptides has been used for many years and has become a common way to biochemically tag targets of interest (1). One advantage is the very strong binding interaction with avidin that can be used to purify biotin-tagged proteins (2,3). It can also be used as a method for conjugation of a variety of tags such as dyes, beads and other solid substrates for a variety of assays and tools (4,5).
New assays have been developed that improve elucidation of protein subcellular context, localization, and protein-protein interactions. These new cellular assays utilize biotin ligase or engineered ascorbate peroxidase (APEX) fusion proteins that are able to biotinylate nearby protein targets within specific cellular compartments. This type of biotin proximity assay can provide improved information about subcellular localization, multi-subunit components, and recruitment of unknown targets. Using CST's in house developed anti-biotin rabbit monoclonal antibody; we have developed a robust procedure for immunoaffinity enrichment followed by mass spectrometry analysis using CST’s proprietary PTMScan(R) protocols. This allows for identification of biotinylated targets and investigation of new protein interactions. This assay provides rich data for these types of biotin proximity assays such as the APEX (6,7) engineered cell system and other related methods.
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