The BIS-300 Kit contains a fully biotinylated MBP-AviTag™ fusion protein standard and an unbiotinylated MBP-AviTag™ fusion protein that may be used as an extent-of-biotinylation comparison for BirA biotin-protein ligase reactions, for SDS-PAGE gel analysis or in Western blot analysis.
Kit supplied with the following:
- One (1) vial containing 50µL of fully biotinylated MBP-AviTag™ fusion protein at 1mg/mL (50µg).
- One (1) vial containing 100µL of unbiotinylated MBP-AviTag™ fusion protein at 1mg/mL (100µg).
By comparing a known amount of a 100% biotinylated AviTag™ fusion protein as a standard against a known amount of BirA-reacted protein-of-interest in an ELISA-type, multi-well dilution format (called BRTA for Biotinylation Reaction Titration Assay) it is possible to assess the extent of biotinylation achieved in your own BirA biotinylation reactions in a highly accurate and reproducible manner. In addition, a mass shift between biotinylated and unbiotinylated fusion proteins visible on both SDS-PAGE and Western blot can be used to subjectively determine the amount of biotinylation that has occurred in a reaction by the observation of differences in band density in a mixed population of biotinylated vs. nonbiotinylated substrate. The BIS-300 Kit contains the fully biotinylated standard used for the BRTA protocol and an unbiotinylated AviTag’d protein to react in a parallel to your BirA/protein biotinylation reaction as a positive control.The BRTA protocol can be found in our protocol section.
How can I quantitate the level of biotinylation of my protein?
We have developed a simple 'dot blot' assay and plate assay