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Protein Site Specific Labeling
We provide custom production of site specific labeled proteins and assay development based on the technology developed in EZBiolab’s research lab.
For more information and to discuss your project, please email us: service@ezbiolab.com
Site is Important
A large number experiments in bioresearch and drug discovery require proteins to be labeled by fluorescence, biotin, phosphorylation or others. The labeling groups are usually conjugated to a protein non-specifically, producing randomly labeled samples. As the results, the site and the number of the labeled groups on the target protein is neither predictable nor controllable. This often disrupts the function of the target protein and produces poor batch-to-batch consistency. For example, in FRET binding assay, if a binding-site residue happens being labeled by a fluorescence or its quench molecule, which often occurs because the binding site is always located on the surface of protein, the binding function can be disrupted. The similar case is also frequently seen in in Biacore binding experiment. This has become a major cause of inconsistent binding affinity reported by different researchers.
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Effects of random labeling and site-specific labeling on binding Assays
Applications of Protein Site-Specific Labeling

1. Enzymatic assays
    With our site-specific labeling technology, the full length natural protein substrates are now available for protease,     phosphatase and some other enzymes to replace their short-peptide substrates
       
  •       • Highly specific reaction
  •       • Full interaction between substrate and enzyme
  •       • Inhibitors bind to the substrate protein to avoid the selectivity problem of active site inhibitors
          Protease
         A fluorescence based protease assay to cleave its natural protein substrate

Example:
Cleavage of pro-TNF alpha by ADAM 17 (TACE): a fluorescence-based protease assay cleaves its natural protein substrate

Anal Biochem. 2014 Jan 15;445:14-9.
           Phosphatase
         The native protein substrate phosphorylated at the exact residue that is hydrolyzed by the targeted          phosphatase
Phosphatase is an important class of drug target. Selectivity has long been a major problem for phosphatase inhibitors. The site-specific phosphorylated protein substrate can help to solve this problem
          Epigenetic Related Enzymes
         Histone deacetylases, protein methyltransferases and lysine demethylases, they all
         catalyze the reactions with site-specific modified protein substrates
2. Binding Assay
    Site specific labeled proteins show un-interfered and consistent binding results in either fluorescence or Biacore     binding assay
Example:
FRET binding assay of site specific labeled fluorescence/quencher pair
Protein Y binds to fluorescence B to form a binding complex. In this assay, protein Y was labeled by a fluorescence dye (5’TAMRA) and protein B was labeled by its quencher (QSY9), respectively, in the site-specific manner. Titration of Y by B results decrease of fluorescence due to the binding.

 

3. Structure and Function Studies of Post-Translational Modified Proteins
    Most post-translational modified proteins such phosphorylated, acylated or myristoylated proteins are difficult to     be purified from natural or cultured cells because of their extremely rare abundance and high heterogeneity.

    With our site-specific labeling technology, those proteins can now be obtained in large quantity for structure and     function studies.

Please contact us for our services:

  •    Custom production of site specific labeled proteins
          Almost every available labeling/modification groups can be site-specifically incorporated into a protein
    •          • Fluorescence
    •          • Biotin
    •          • Phosphorylation
    •          • Acylation
    •          • Methylation
    •          • Non-natural amino acids
    •          • Stable isotope
    •          • Myristoylation
    •          • Palmitoylation
    •          • More


  •    Assay development using site specific labeled proteins
    •          • Binding assay
    •          • Protease assay
    •          • Phosphatase assay
    •          • Other assays
For more information and to discuss your project, please email us: service@ezbiolab.com